NFGEL 2000-2001 Annual Report
Annual Report
2000-2001(FY01)
Table of Contents
Introduction
Projects
Overview
Silviculture and Tree Improvement
Ramet/Genet Identification in Port-Orford
Cedar (Chamaecyparis lawsoniana)
Genetic Variation and Hybridization in
Cupressus, Chamaecyparis, and X
Cupressocyparis
Efficacy of Supplemental Mass Pollination
Methods In A Douglas-Fir (Pseudotsuga
menziesii) Seed Orchard
Effects of Stand Density Reduction
Treatments On the Genetic Variation of
Ponderosa Pine (Pinus Pondreosa) In
Northern Arizona
Clonal Identification in Douglas-fir
(Pseudotsuga menziesii)
Cross Verification Among Chamaecyparis
Species
Conservation and Restoration
Genetic Diversity In Perideridia
erythrorhiza: A Rare Plant In Southern
Oregon
Genetic Differentiation In A Rare Plant With
A Disjunct Range: Lewisia kelloggii
Genetic diversity in Broadleaf Lupine
(Lupinus latifolius) accessions from the Mt.
Hood National Forest
Staff Activities
Lab Management
Staffing
Budget
USDA Forest Service -NFGEL, 2480 Carson Road,
Placerville, CA 95667
530-622-1609 (office), 530-622-1225 (lab)
530-622-2633 (fax)
Lab Workload
By Project
By Region or Agency
Prior Annual Reports
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NFGEL 2000-2001 Annual Report
FY98
FY99
FY00
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NFGEL INTRODUCTION 2000-2001
INTRODUCTION
After residing for 13 years on the Eldorado National Forest, NFGEL has relocated to the Pacific
Southwest Research Station, Institute of Forest Genetics, Placerville CA. Many thanks go to the
NFGEL staff for their dedicated efforts in making this move happen. A move of this magnitude
required both physical and mental energy - both of which they showed in full measure. It took only two
weeks in our new home to be fully up-and-running, producing results of the high quality that are
standard to our procedures. Thanks also go to the IFG scientists and staff who have been enormously
welcoming and accommodating. It is also through their efforts that our transition has been so seamless.
Everyone who has been involved in this move, at the local, Regional, Station, and Washington Office
levels, deserves our sincere thanks.
NFGEL remains a National Forest System facility. With this move, we plan on strengthening our
partnerships with the Forest Service Research branch, and improving our role of 'linking science to
management'. We were able to add some new equipment this fiscal year that will enable us to extend
the services we provide. Our most promising purchase was of an ABI-3100. We anticipate generating
highly variable DNA markers and sequence data that can be used in the assessment of many of the
plant species we study.
As can be seen in this report, the scope of our work continues to grow. At the end of this report period,
we had accepted our 125th project. We look forward to continuing our work in support of genetic
improvement and conservation efforts in our new home at the Institute of Forest Genetics.
Valerie Hipkins
NFGEL Director
October 2001
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NFGEL Overview 2000-2001
Overview
During this FY01 report year, we processed 6 projects utilizing isozyme markers obtained by
starch gel electrophoresis, performed development work on 8 other isozyme projects, and initiated
work on 11 additional projects using DNA markers. NFGEL projects were processed to meet a
variety of management objectives. Nine reports, including results from 12 projects, follow.
Silviculture and Tree Improvement
1. Ramet/Genet Identification in Port-Orford Cedar (Chamaecyparis lawsoniana)
2. Genetic Variation and Hybridization in Cupressus, Chamaecyparis, and X
Cupressocyparis
3. Efficacy Of Supplemental Mass Pollination Methods In A Douglas-Fir
(Pseudotsuga menziesii) Seed Orchard
4. Effects Of Stand Density Reduction Treatments On The Genetic Variation Of
Ponderosa Pine (Pinus Ponderosa) In Northern Arizona
5. Clonal Identification in Douglas-fir (Pseudotsuga menziesii)
6. Cross Verification Among Chamaecyparis Species
Conservation and Restoration
1. Genetic Diversity In Perideridia erythrorhiza: A Rare Plant In Southern Oregon
2. Genetic Differentiation In A Rare Plant With A Disjunct Range: Lewisia kelloggii
3. Genetic Diversity In Broadleaf Lupine (Lupinus latifolius) Accessions From The
Mt. Hood National Forest
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P97 Report
Ramet/Genet Identification in Port-Orford Cedar (Chamaecyparis
lawsoniana)
Objective
The project objective was to verify that ramets of each clone are identical, but that clones are unique.
Materials
Twenty individuals of Port-Orford Cedar (Chamaecyparis lawsoniana) (POC), putatively labeled as
eight different clones (one to six ramets per clone) were submitted for analysis.
Analysis
Individuals were prepared for both isozyme (starch gel electrophoresis) and RAPD (random amplified
polymorphic DNA) analysis. Sample preparation for starch gel electrophoresis followed NFGEL Standard
Operating Procedures. Genomic DNA was extracted by a modified Jorgenson CTAB protocol and yielded
between 0.5 - 3ug of DNA per sample.
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Starch Gel Electrophoresis. Individuals were genotyped at 22 isozyme loci (FEST-1, PGM-1, ME7,
PGI-1, PGI-2, UGPP-1, TPI-1, G6PD, GOT-1, GOT-2, GDH, MDH-1, MDH-2, 6PGD-1, 6PGD-2, IDH,
SKD-2, FDP-1, SOD-1, SOD-2, ACP, MNR). The multilocus genotype data show that all ramets of each
genotype are identical. However, isozyme data did not distinguish all genotypes. Clone 510015 and clone
CF01 share the same multilocus genotype. Clones CF02, CF03, and CF06 share an identical isozyme
genotype. The remainder of the clones have unique genotypes. Overall, genetic variation among genotypes
is low with 82.6% of the loci monomorphic. The loci that distinguish clones are FEST1, PGI2, TPI1 and
GDH.
RAPD Analysis. In order to further distinguish groups 510015/CF01 and CF02/CF03/CF06, one
individual per putative clone was characterized using RAPD markers. One individual per clone was used
in the analysis based on the assumption that all ramets of each genotype are identical (as indicated by the
isozyme analysis). Therefore, RAPD markers were generated for five individuals (510015, and one
individual from each of CF01, CF02, CF03, and CF06) using 45 RAPD primers. Reaction samples
contained 1.25mM dNTPs, 0.4uM primer, 1U Taq, and 3 ng DNA in a 25ul volume. Thermal steps
consisted of forty cycles of one minute denaturation at 94o, one minute annealing at 40 o, and 2 minute
extension at 72o. Entire reaction volumes were loaded onto 1.4% agarose gels in 1X TBE. Data was
scored with arbitrary numbers representing the overall band pattern.
Generally, the RAPD patterns generated for 510015 and CF01 were similar to each other. Likewise, the
pattern generated for CF02, CF03, and CF06 were similar to each other. The 510015/CF01 group varied
distinctly from the CF02/CF03/CF06 group (Figure 1).
Although there were similarities within the groups, clones could be distinguished as unique. RAPD
markers clearly distinguished clone 510015 from clone CF01 at 26 primers (see Figure 2 for example).
RAPD markers distinguished clone CF03 from CF02/CF06 at 16 primers (see Figure 3 for example).
Clones CF02 and CF06 were not easily distinguished and clearly differed at only one of the 45 primers
tested (Figure 3). The bands obtained with the OPB-03 primer were reproducible. Four other primers
(OPA-19, OPB-02, OPB-18, and OPH-11), also showed differences between CF02 and CF06, but these
differences were not clear.
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Conclusion. All ramets of each submitted clone are identical. Clones 117490, 510015, CF01, CF03,
CF08, and CF27 are unique. Although clones CF02 and CF06 could only clearly be distinguished with one
RAPD primer, we conclude that they are also distinct clones.
This was part of NFGEL Project #97
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Genetic Variation and Hybridization in Cupressus, Chamaecyparis, and X
Cupressocyparis
INTRODUCTION
We used a variety of molecular techniques to test the hypothesis that several submitted individuals
were hybrids between either:
1. Cupressus macrocarpa (Monterey cypress) X Chamaecyparis lawsoniana
(Port-Orford cedar) (POC),
2. Chamaecyparis lawsoniana X Chamaecyparis nootkatensis(Alaska yellow
cedar)(AYC), or
3. Cupressus macrocarpa X Chamaecyparis nootkatensis.
Levels of genetic variation within and among species was also assessed.
Molecular genetic data have been successfully used to address the question of interspecific
hybridization in other species. As long as the parent species are sufficiently divergent genetically at the
time of the hybridization event, the hybrid should show additivity of the genetic markers to the parent
species. Unique bands can also be expected to be rare in the hybrid.
METHODS AND MATERIALS
A total of 38 individuals were used to assess intraspecific variation within POC. Collections were
submitted by Rod Stevens, BLM (17 samples), and Rich Sniezko/Leslie Elliot, Dorena Genetic
Resource Center, USDA Forest Service (21 samples). Dorena submissions included rangewide
samples.
Twelve individuals, ten submitted by BLM and two submitted by Dorena, were used to assess
variation in AYC. Thirty-five putative AYC X POC hybrids were included for testing (from BLM). A
total of seven individuals of Monterey cypress were submitted from the BLM.
Also included for testing were four individuals of Chamaecyparis obtusa (two from BLM, two
from Dorena), one individual of Chamaecyparis thyoides from Dorena, two individuals of Cupressus
torulosa (from BLM), and five individuals of Leyland cypress (Cupressus macrocarpa X
Chamaecyparis nootkatensis) (from BLM). Multiple collections of a putative Monterey cypress X
POC hybrid were submitted.
Starch Gel Electrophoresis
A small section of needle tissue (~3 mm3) per tree was placed in a microtiter plate well containing
150ul of 'Melody/Neale' extraction buffer. The plate was frozen at -70C. On the morning of the
electrophoretic run, the samples were thawed, macerated with a dremel tool, and the extract absorbed
onto three, 3mm wicks. Sample wicks were loaded into 11% starch gels that accommodated 30
samples along the longitudinal axis.
The following enzymes were examined: fluorescent esterase (FEST), phosphoglucomutase (PGM),
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phosphoglucose isomerase (PGI), malic enzyme (ME), phosphogluconate dehydrogenase (6PGD),
triosephosphate isomerase (TPI), uridine diphosphoglucose pyrophosphorylase (UGPP),
glucose-6-phosphate dehydrogenase (G6PDH), glutamate-oxaloacetate transaminase (GOT), glucose
dehydrogenase (GDH), fructose-1,6-diphosphate (FDP), shikimic acid dehydrogenase (SKD), malate
dehydrogenase (MDH), and isocitrate dehydrogenase (IDH). A lithium borate electrode buffer (pH
8.3) was used with a Tris citrate gel buffer (pH 8.3) to resolve PGM-1, PGI-1,2, ME7, and FEST-1. A
sodium borate electrode buffer (pH 8.0) was used with a Tris citrate gel buffer (pH 8.8) to resolve
TPI-1, UGPP-1, G6PD, GOT-1,2, and GDH. A morpholine citrate electrode and gel buffer (pH 8.0)
was used to resolve MDH-1,2, 6PGD-1,2, SKD-2, FDP-1, and IDH. Two people independently scored
each gel. When they disagreed, a third person resolved the conflict. For quality control, 10% of the
individuals were run and scored twice.
Isoelectric Focusing
Three hundred milligrams of needle tissue per tree was ground in a cold mortar containing 2ml of a
glycine extraction buffer. Extracted samples, including liquid and solid materials, were stored in 5ml
cryovials at -20C until electrophoresis.
On the day of electrophoresis, samples were thawed and loaded onto polyacrylamide gels, type
FS5080 (pH 4-5). Gels were stained for EST, PER, PHI, MDH, DIA, ACP, and PGM.
Random Amplified Polymorphic DNA (RAPD)
DNA extractions were carried out using a modified FastPrep protocol (Bio101 Inc.). DNA was
quantified by specific fluorescence detection in a Hoeffer Scientific fluorometer. Based on
quantification, samples were diluted to 3 ng/ul for PCR amplifications. On some samples, DNA was
quantified using a DNA Dipstick (Invitrogen).
PCR reaction mixtures (25ul) contained 0.5 uM of a 10-base primer (Operon Technologies Inc.), 3
ng of genomic DNA, and 1 unit Taq DNA polymerase. A DNA thermal cycler (MJ Research) was
programmed for 2 min at 94C followed by 40 cycles of 1 min at 94C, 1 min at 40C, and 2 min at 72C
to carry out the amplification reactions. A final step of 10 min at 72C was added at the end of the
cycle. RAPD products were analyzed by electrophoresis on 1.4% agarose gels in 1X TBE and stained
in ethidium bromide. Gels were photographed under transmitted UV light using a Polaroid camera.
Scoring was performed directly from the photographs.
RESULTS AND DISCUSSION
VARIATION WITHIN SPECIES
The genetic variation found in POC (as measured by starch gel electrophoresis) is lower than the
average gymnosperm, and only slightly lower than that of the average long-lived woody species with a
regional distribution (see Table). Alaska yellow cedar contained greater levels of diversity than
Port-orford cedar. The genetic variation detected in Monterey cypress is very low compared to the
average gymnosperm. Other species analyzed had such small sample sizes that within species diversity
level statistics are not meaningful.
Of the seven Monterey cypress individuals analyzed, five of them had the same multilocus
genotype. The 'Jam & Jelly - mature' sample had a unique genotype as did the 'Jam & Jelly - seedling'
sample. The 'mature' and 'seedling' samples did not match each other. The five samples may have
matching genotypes because (1) they are the same genetic individual, (2) there is little variation in
Monterey cypress, or (3) there is little variation among the five samples that happened to have been
chosen for analysis. A larger sampling of Monterey cypress would address this question. .
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Table. Levels of genetic variation within taxa. Standard errors in parenthesis.
Taxon
Average gymnosperm
Ave. long-lived woody
species with regional
distribution
Port-Orford Cedar
Alaska yellow cedar
Monterey cypress
Percentage of
Mean sample size
Mean # of
per locus
alleles per locus polymorphic loci
(P)
(A)
-2.38 (0.1)
71.1 (2.6)
-1.87 (0.1)
55.7 (2.3)
Expected
heterozygosity
(He)
0.169 (0.008)
0.169 (0.008)
11.8 (0.2)
11.8 (0.2)
1.8 (0.2)
1.8 (0.2)
55.6
55.6
0.131 (0.038)
0.204 (0.054)
6.8 (0.2)
1.3 (0.1)
22.2
0.083 (0.044)
VARIATION AMONG SPECIES
There are many distinct genetic differences among the taxa studied. POC and AYC share only
33.7% genetic similarity. POC is only slightly more similar to Monterey cypress (37.8%) than it is to
AYC. Monterey cypress is more similar to AYC (65.6% genetic similarity) than POC is to AYC
(which is suprising given that Monterey cypress is in the genus Cupressus, while POC and AYC are in
the genus Chamaecyparis). In the following dendrogram, the Cupresses species form a branch than
includes AYC. Ch. obtusa is distinct from the other species, as is the Ch. thyoides. Cupressus torulosa
shares over 74% genetic simarity to Monterey cypress, slightly greater than expected.
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Six loci (out of 18 assessed) show fixed differences between POC and AYC. There are 35 allelic
differences between the species (an allele that occurs in one species and not the other). There are five
fixed differences (33 allelic differences) between POC and Monterey cypress. Comparing Monterey
cypress to AYC, only two fixed differences and 26 allelic differences distinguish the species.
TESTING THE HYBRID HYPOTHESIS
POC X AYC
The putative POC X AYC hybrids appear to be either POC or AYC - not hybrids between the two
species. All three molecular techniques used (starch gel electrophoresis, isoelectric focusing, and
RAPDs) indicated that putative hybrids were either members of one species or the other. This was
determined by starch gel electrophoresis at 18 isozyme loci, isoelectric focusing at three isozyme loci,
and RAPD data at 171 band markers generated from 22 primers.
Individuals '11311B', '11312A', and 'number 01' appear to be AYC. This group of three
individuals are referred to as 'AYC-unk'. All other submitted putative POC X AYC hybrids appear to
be POC (this groups of 32 individuals is referred to as 'POC-unk'). POC and POC-unk share over 99%
genetic similarity. AYC and AYC-unk share 99.8% similarity. The putative hybrids are not showing
additivity of the genetic markers to the parent species, as would be expected in a hybrid. In fact, the
AYC-unk group shares multiple markers with AYC that don't exist in POC. AYC-unk shares no
markers with POC that don't also exist in AYC. This same pattern of non-additivity is true for the
POC-unk group. This group shares many markers with POC that don't exist in AYC. POC-unk shares
no marker with AYC that doesn't also exist in POC.
Leyland cypress (X Cupressocyparis leylandii)
Leyland cypress is a hybrid between Monterey cypress and Alaska yellow cedar. Five individuals
of Leyland cypress were genetically analyzed: BLM#1, Castlewellan, Green, Naylor Blue, and Silver
Dust. Naylor Blue showed no hybrid pattern between Monterey cypress and AYC. It instead showed a
high degree of similarity to Port-orford cedar (around 85% genetic similarity). Naylor Blue showed
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only 35% similarity to AYC, and 29% similarity to Monterey cypress. Because it appears that Naylor
Blue is either a Port-orford cedar (see preceding dendogram), or at least a cross involving Port-orford
cedar, it was removed from the Leyland cypress group for the remaining analysis.
Of the remaining four individuals of Leyland cypress, three of them have matching genotypes:
BLM#1, Green, and Silver Dust. It is possible that these individuals are really ramets of the same
clone, or they just happen to have matching genotypes. RAPD data was not generated on the Leyland
cypress individuals. DNA was extracted and stored, so RAPDs could be generated if needed.
Castlewellan has a unique genotype.
The Leyland cypress group is most genetically similar to the putative Monterey cypress X POC
hybrid (97.7%), the Monterey cypress (91.1%), and the Alaska yellow cedar (70.7%). So even though
the Leyland cypress is more similar to the Monterey cypress than the AYC, it does show additivity of
the genetic markers between both parents. Leyland cypress shares four alleles with Monterey cypress
that do not exist in AYC; it also shares four different alleles with AYC that do not exist in Monterey
cypress. Its position on the dendogram also diagrams its relationship to both Monterey cypress and
AYC.
Monterey cypress X Port-Orford cedar
Several individuals of a putative Monterey cypress X POC cross where submitted. These included
samples 60886, 60887, 61326, and 'PO x C. macrocarpa'. In previous NFGEL results, we reported that
all four samples had identical multilocus genotypes. We also stated that "It is likely that these are
ramets of a single clone as opposed to separate individuals of the same controlled cross". The hybrid
('MC X POC') is very similar to that of Monterey cypress, and distinct from POC. 'MC X POC' shares
97.5% similarity to Monterey cypress, 70.9% similarity to Alaska yellow cedar, and only 34.7%
similarity to POC. Interestingly, 'MC X POC' is most similar to Leyland cypress (excluding Naylor
Blue). Leyland cypress and 'MC X POC' share 97.7% similarity. 'MC X POC' also shows some
additivity of marker bands to Monterey cypress and AYC. Based on these genetic results, it is clear
that the putative 'MC X POC' hybrid involves Monterey cypress. It either is a pure Monterey cypress
or is the result of a cross with Monterey cypress. The data do suggest that the putative hybrid could be
a cross between Montery cypress and AYC, not POC. This is supported by (1) the putative hybrid
showing additivity of genetic markers to Monterey cypress and AYC, (2) the high genetic similarity
(relative to other taxa) between the putative hybrid and both AYC and, especially, Monterey cypress,
and (3) the placement of the putative hybrid with Leyland cypress in the dendrogram.
CONCLUSIONS
●
●
●
●
●
Monterey cypress and Port-Orford cedar contain low levels of genetic diversity compared to the
average gymnosperm.
Alaska yellow cedar contains comparable (to slightly low) levels of genetic diversity compared
to the average gymnosperm.
The Chamaecyparis and Cupressus species studied are significantly divergent. They clearly
show many genetic differences, both fixed differences and differences in marker frequencies. (It
should be noted that several species were represented by only one to four individuals).
All putative Port-Orford cedar X Alaska yellow cedar hybrids appear to be one species or the
other, not hybrids between the species.
Leyland cypress does appear to be a hybrid between Monterey cypress and Alaska yellow cedar
(except for Leyland cypress-NaylorBlue which appears to be Port-Orford cedar, or from a cross
involving Port-Orford cedar).
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We could not definitively identify the parents of the putative Monterey cypress X Port-Orford
cedar hybrid. The hybrid clearly involves Monterey cypress as a parent (it is either pure
Monterey cypress or is the result of a cross with Monterey cypress). If this material is a hybrid,
the data suggest a higher probability that the other parent is Alaska yellow cedar instead of
Port-Orford cedar. However, the data cannot rule out the possibility that the material is a
Monterey cypress X Port-Orford cedar hybrid.
● It should be noted that molecular genetic evidence of hybridization can be misleading if it is
used to assess advanced generation hybrids.
This was NFGEL Project #s 86, 87, 88 and part of 97.
●
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P94 Douglas-fir
Efficacy Of Supplemental Mass Pollination Methods In A Douglas-fir Seed
Orchard
The goal of this project was to determine the efficacy of supplemental mass pollination methods
in a privately-owned Douglas-fir seed orchard by genotyping megagametophyte and embryo (m/e)
pairs from four controlled cross and two open-pollinated cross seedlots. Controlled crosses consisted
of two different females each crossed with two different males. Open-pollinated seed was also
collected from each female.
Seed was prepared and electrophoresed following NFGEL Standard Operating Procedures. We
genotyped a total of 328 seed (m/e pairs) at 15 isozyme loci using three buffer systems via starch gel
electrophoresis. Between 26 and 104 seed per cross was genotyped (sample size depended on seed
availability - 100 seed per cross was the target).
Contamination levels in crosses using 'male #1' was low (four seed out of 104 (3.8%), and two
seed out of 26 (7.7%) were detected contaminants). The crosses using 'male #2' showed high levels of
contamination (67.3% and 30.9%). There seemed to be substantial pollen from an unknown male
mixed with that from 'male #2'. The open-pollinated crosses were fairly diverse.
This is NFGEL Project #94.
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PJ99 Ponderosa Pine
Effects Of Stand Density Reduction Treatments On The Genetic Variation
Of Ponderosa Pine In Northern Arizona
By: Kristin Kolanowski
The purpose of the research is to determine if the genetic variation of Pinus ponderosa var.
scopulorum in Flagstaff's urban wildland interface (FUWI) has been affected by changes in
composition, structure, and function that have taken place in the forests over the last 130 years. The
research will also address the importance of including genetic considerations in resource management
by quantifying the effects of different simulated thinning treatments, based on full restoration
guidelines, on the genetic variation of ponderosa pine in FUWI.
The objectives of the research are to determine the genetic variation partitioning of clumps of
trees that established prior to Euro-American settlement (1876), determine the genetic composition
and structure of pre- and post-settlement trees, compare allozyme variation of the two age groups, and
compare the genetic composition and structure of 5 stands prior to and after simulated random
thinnings having 50, 25, and 10% post-settlement retaining percentages. The following hypotheses
will be tested:
1. There is more within-clump variation than among-clump variation of
pre-settlement trees.
2. The genetic variation of pre-settlement trees is different from that of
post-settlement trees.
3. The genetic variation of spatially selected (replacement) post-settlement trees does
not differ from that of randomly selected post-settlement trees.
4. A thinned stand will have less genetic variation (less heterozygosity, smaller % of
polymorphic loci, mean # of alleles per locus, etc.) than that of the same stand that
has not been thinned.
The goal and final step will be to suggest how to incorporate genetic guidelines resulting from the
research into management of ponderosa pine ecosystems in northern Arizona.
A total of 465 ponderosa pine trees were genotyped at NFGEL using vegetative bud tissue. Data
was obtained at 22 isozyme loci using three buffer systems. This information is part of a Master's
Degree program by the author at Northern Arizona University in Flagstaff, Arizona. Analysis and
reporting are in progress.
This is NFGEL Project #99.
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P112 Douglas-fir
Clonal Identification in Douglas-fir (Pseudotsuga menziesii)
The objective of this project was to perform clonal and parental identification in a Douglas-fir
(Pseudotsuga menziesii) seed orchard operated by a private company out of Washington. Branch tips,
including needles and expanding buds, from 44 individuals of Douglas-fir were submitted for analysis.
Between two and five ramets were provided for 10 different clones. Eight open-pollinated progeny
were provided from an eleventh clone, along with two possible mother trees (both labeled as ramets of
the same clone).
Vegetative bud material from each tree was prepared for analysis following NFGEL Standard
Operating Procedures. Material was electrophoresed on 11% starch gels and assayed at 17 isozyme
loci using three buffer systems.
Isozyme data showed that all ramets of eight of the clones were correctly identified. Some ramet
mislabeling was found in the other two clones. Progeny individuals were identified as offspring of one
of the two submitted putative mother trees.
This was NFGEL Project #112.
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P102 Douglas-fir
Cross Verification Among Chamaecyparis Species
The project objective was to verify hybrid seedlings resulting from seven crosses between
Chamaecyparis lawsoniana (Port-Orford Cedar, POC), Chamaecyparis obtusa(CHOB),
Chamaecyparis nootkatensis (Alaskan Yellow Cedar, AYC), and Chamaecyparis thyoides(CHTH).
METHODS
Branch tips from 52 individuals were submitted for analysis. A small section of needle tissue (~3
mm3) per tree was placed in a microtiter plate well containing three drops of Gottlieb extraction buffer
(NFGEL Standard Operating Procedures). One replicate plate was made of each set. Plates were
frozen at -70C. On the morning of the run, samples were thawedd, macerated with a dremel tool, and
the extract absorbed onto three, 3mm wicks. Samples were genotyped at 19 isozyme loci.
RESULTS
POC #1 x CHOB #2 Seedling '144-1' appears to be the product of a successful cross between these
two parents. Seedlings '144-2' and '144-3' are not progeny from a cross between these two parents.
POC #2 x CHOB #2 Seedling '145-4' appears to be the product of a successful cross between these
two parents. The remaining seedlings in this group are not progeny from a cross between these two
parents.
POC #3 x CHOB #1 None of the seedlings could have been produced from a cross between these two
parents. The seedlings are also not from a 117335 self cross (though the paternal parent for all is likely
a Port-Orford Cedar(s)).
CHTH x self All seedlings appear to be the result of a successful self cross.
CHTH x POC #3 None of the seedlings could have been produced from a cross between these two
parents. The seedlings could have been produced from a bluesport self cross. No Port-Orford Cedar
appears to have served as a pollen parent for these seedlings.
CHOB #3 x POC #3 None of the seedlings could have been produced from a cross between these two
parents. The seedlings could have been produced from a CHOB gracilis self cross. No Port-Orford
Cedar appears to have served as a pollen parent for these seedlings.
POC #4 x AYC Because no parental material was sent for genotyping, verification of the cross is not
possible. However, it is likely that the cross was not successful. Based on prior genotyping of
Port-Orford Cedar and Alaska Yellow Cedar (AYC), these seedlings have none of the alleles typical of
those found in AYC. This project was done in cooperation with Richard Sniezko, USDA Forest
Service, Dorena Tree Improvement Center, Cottage Grove, OR 97424, and is NFGEL Project #102.
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Genetic Diversity In Perideridia Erythrorhiza: A Rare Plant In Southern
Oregon
Study Objectives and Summary
1a. Do the populations from the three general locations (Klamath Falls,
Junction) differ on a genetic basis?
Roseburg, and Cave
Yes, they do differ. However, the populations within the regions are more highly differentiated
than the regions are. The Cave Junction region is the most genetically divergent of the general
locations. Klamath and Roseburg locations are more similar to each other than either is to Cave
Junction. The Klamath location shares 82.9% genetic similarity with Roseburg (mean genetic
identities among conspecific populations are usually high (above 90%); among congeneric
species are usually low (below 70%); and among infraspecific taxa are usually intermediate to
the conspecific and congeneric values: although it should be noted that these are mean values,
and individual situations vary greatly).
1b. If the populations/locations do differ on a genetic basis, do the
differences warrant separation into different species?
The isozyme variation in itself does not warrant separation of the general locations into
separate species. The great differentiation among populations might allow separation on the
basis of other
(non-isozyme) evidence.
The Leather population from the Cave Junction location does not appear to be Perideridia
erythrorhiza. It is either a mis-identified collection of another species, or a new species. The
Leather population should be re-examined. That examination should include both the collection
of herbarium voucher specimens and isozyme analysis of a second seed collection, to be sure
the vouchers match the isozyme observations. If isozyme variation in Perideridia erythrorhiza
is studied further, we recommend that at least one additional population from the Illinois River
area be included.
1c. Are there genetic differences between the eastern and western
populations (east or west of the Cascades)?
It has been hypothesized that variation in P. erythrorhiza can best be divided into two groups,
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an eastern group (including populations Howard Prairie, Mud Flat, and Pelican Barn) and a
western group (including populations Illinois River, Roseburg, and Umpqua). We did not find
that division to best express the pattern of isozyme variation we observed. A more useful way
to express the pattern of variation is to divide the populations into three regions; Roseburg (the
Roseburg and Umpqua populations), Cave Junction (the Illinois River population), and
Klamath Falls (the Howard Prairie, Mud Flat, and Pelican Barn populations).
2. Does the Pelican Barn population from the Winema National Forest differ
from the population at Mud Flat?
The Pelican Barn and Mud Flat populations are not identical, but their genetic identity (90%) is
the fourth highest observed between any pair of populations. The data indicate that there might
be some inbreeding, selfing, or population substructuring in the Pelican Barn population that is
not occurring in the Mud Flat population. Mud Flat shares 99.7% similarity with the Howard
Prairie population.
3. Is the genetic diversity in the Pelican Barn population substantially smaller
than the diversity in the Mud Flat population?
The Pelican Barn population was slightly less variable than the Mud Flat population in all
diversity measures except for expected heterozygosity. If all diversity statistics are weighted
equally, the Illinois River population is the most diverse, followed by, in descending order of
diversity level, Mud Flat, Roseburg, Pelican Barn, Howard Prairie, and Umpqua.
INTRODUCTION
Perideridia erythrorhiza (Piper) Chuang & Constance is a rare perennial herb that occurs in southern
Oregon in only three general locations: near Klamath Falls in Klamath and Jackson Counties, near Roseburg
in Douglas County, and near Cave Junction in Josephine County. This plant is commonly known as
red-rooted yampah and is related to parsnips and carrots in the family Apiaceae. This species is considered
approximately tetraploid, with n = 19 (Chuang and Constance 1969). It is thought to be outcrossing, though
capable of self-pollinating (Meinke 1998). The biology, history, and geographic range of the species are
covered elsewhere (Chuang and Constance 1969, and Meinke 1998). Perideridia erythrorhiza is classified
by the US Fish and Wildlife Service as a candidate threatened species, and by the USDA Forest Service
Region 6 as a sensitive species.
Perideridia erythrorhiza occurs in two locations on the Klamath Ranger District of the Winema National
Forest, USDA Forest Service (Klamath Fall area, Klamath County). These populations (Pelican Barn and
Odessa sites) are small and not very robust. However, these populations are important for the conservation
of the species because most other populations in this area are on private land or are threatened by
development or recreational use. A third population located in the Klamath Falls area occurs in Jackson
County and is located at Mud Flat on industrial land that is both grazed and logged. The Mud Flat
population is the largest and most vigorous population known in the entire species. This population is an
obvious choice of plants to augment the smaller populations on Forest Service land if mitigation became
necessary. The Mud Flat site is separated from the Klamath County sites by 25 miles. F1
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Figure 1. Locations of Perideridia erythrorhiza populations sampled for the genetic study.
The Forest Service is interested in obtaining genetic data to understand the taxonomic relationships
among the general locations with the species (Figure 1), and also to determine the levels of diversity within
and among the general locations and populations with a location. Specific objectives of the study include:
(1) determine if populations from the three general locations (Klamath Falls, Roseburg, and Cave Junction)
differ on a genetic basis, and if so, do the differences warrant separation into different species; (2) determine
if the Pelican Barn population from the Winema National Forest differs from the population at Mud Flat;
and (3) determine if the genetic diversity in the Pelican Barn population on the Winema NF is substantially
smaller than the diversity in the Mud Flat population.
MATERIAL AND METHODS
Sample Collection. Seed was collected from between six and sixty-one individuals per population.
Three to five seed per plant were stratified and germinated. The collection, stratification, and germination of
seed was performed by Paul Berrang, USDA Forest Service. Once seedlings grew to where their shoots
were approximately 5 - 10 cm in length, they were transferred to NFGEL for genetic analysis. In addition, a
bulk seed sample from 75 - 100 plants was collected at the Illinois River population by Kim Roberts.
Seedlings were grown and processed like those from the other populations, but results were omitted from
most analysis; see Results and Discussion.
Sample Preparation. Seedlings from a total of 338 individuals were submitted for isozyme analysis
using starch gel electrophoresis (excluding an Illinois River-bulk collection). One seedling per individual
was prepared for analysis. A pilot study indicated that to obtain adequate isozyme activity, it was necessary
to include the tuberous root in the preparation. Therefore, each seedling was gently dug out of the soil taking
care not to break the shoot off of the tuberous root. Each seedling was washed with water and ground in a
mortar using liquid nitrogen. Approximately 0.4 mls of a modified Pitel and Cheliak (1984) extraction
buffer #7 was added to the ground powder (buffer modifications: 10% PVP-40, 5.0mM EDTA, 10mM DTT,
0.8mM NAD, 0.5mM NADP, buffer pH=8, excluded B-mercaptoethanol). Resulting slurry was transferred
to microtiter plate wells and plates frozen until electrophoresis.
Electrophoresis. In preparation for electrophoresis, slurry was thawed and absorbed onto 3 mm wide
wicks prepared from Whatman 3MM chromatography paper. Methods of electrophoresis followed the
general methodology of Conkle et al. (1982) with some modifications (USDA Forest Service 2000). A
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lithium borate electrode buffer (pH 8.3) with a Tris citrate gel buffer (pH 8.3) (Conkle et al. 1982) was used
to resolve malic enzyme (ME), alcohol dehydrogenase (ADH), phosphoglucomutase (PGM), and
phosphoglucose isomerase (PGI). A sodium borate electrode buffer (pH 8.0) was used with a Tris citrate gel
buffer (pH 8.8) (Conkle et al. 1982) to resolve glutamate-oxaloacetate transaminase (GOT), glucose
dehydrogenase (GDH), triosephosphate isomerase (TPI), and uridine diphosphoglucose pyrophosphorylase
(UGPP). A morpholine citrate electrode and gel buffer (pH 8.0) (USDA Forest Service 2000) was used to
resolve malate dehydrogenase (MDH), phosphogluconate dehydrogenase (6PGD), and fluorescent esterase
(FEST). All enzymes were resolved on 11% starch gels. Stain recipes for enzymes follow USDA Forest
Service (2000). Two people independently scored each gel. When they disagreed, a third person resolved the
conflict. For quality control, 10% of the individuals were run and scored twice.
Data Interpretation. Perideridia erythrorhiza has a haploid chromosome number of n = 19 and is
presumably a tetraploid (Chuang and Constance 1969). Although all loci of a tetraploid should theoretically
contain four alleles, observed band patterns were not consistent with genetic expectations. We therefore
treated the plants as if they were diploid and scored each set of homoeologous loci as one locus. See
discussion for an explanation of this decision.
Genetic interpretations were inferred directly from isozyme phenotypes based on knowledge of the
generally conserved enzyme substructure, compartmentalization, and isozyme number in higher plants
(Gottlieb 1981, 1982; Weeden and Wendel 1989).
Data Analysis. Results were analyzed using Biosys-1, version 1.7 (Swofford and Selander 1989). A
locus was considered polymorphic if an alternate allele occurred even once. We calculated unbiased genetic
distances (Nei 1978), and expected heterozygosity (Nei 1973). F statistics for the hierarchy of populations
within locations, populations within species, and locations with species were calculated by the method of
Wright (1978). Dendrograms based on unbiased genetic distances (Nei 1978) were generated using
UPGMA.
RESULTS AND DISCUSSION
Sampling. An Illinois River-bulk population was omitted from most analysis, for two reasons. First, it
turned out to be a sample from the same population as the other Illinois River sample submitted. Second, its
collection protocol was different from that used for all other populations. The Illinois River-bulk population
was collected from 75-100 female parents and the seed was combined (an unknown number of seed per
parent was collected). All other populations were sampled as individual female plant collections (seed from
each plant was kept separate). Only one seed per plant was genetically analyzed from these populations. A
total of 50 seed was analyzed from the Illinois River-bulk population (however, this could include multiple
seeds from any one plant).
Genetic Interpretation. Perideridia erythrorhiza has a haploid chromosome number of n = 19 and is
presumably a tetraploid (Chuang and Constance 1969). This necessitated considering several options for
scoring isozyme bands. Polyploids often exhibit isozyme band patterns so complex that they defy genetic
interpretation. Such patterns may be treated statistically as phenotypes (Chung et al. 1991, Strefeler et al.
1996). A genetic interpretation of isozyme band pattern data is preferred over a phenotypic analysis, because
a genetic analysis provides more precise information about genetic variation (Gottlieb 1977) and because
results can be compared directly with compilations of plant isozyme genetics (e.g. Hamrick and Godt 1990).
The band patterns in Perideridia erythrorhiza were relatively simple, permitting genetic interpretation.
Once the decision to provide genetic analysis is made, further decisions are required about which loci to
include, treatment of homoeologous loci, and the distribution of alleles among homoeologous loci. Studies
of population genetics in polyploids usually employ one of three approaches to determining the number of
loci scored for each enzyme. (1) Assume that a tetraploid has two homoeologous loci for each enzyme, and
score both of them for every enzyme (Knapp and Rice 1996). This method maximizes the number of
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monomorphic loci and minimizes the number of alleles per locus. Although this is a logical inference from
polyploidy, this approach is rarely used. (2) Assume each enzyme is produced by one locus unless
hypothesizing a second locus is necessary because of fixed heterozygosity or individuals with three or more
alleles per apparent locus (Perez de la Vega 1994, Sanders and Hamrick 1980, Schierebeck et al. 1995). This
method may be chosen because it does not require the inference of "invisible" loci (Nevo et al. 1982), and it
may be the most common way of scoring isozymes in polyploids. It is used "by default" when the
chromosome number is unknown. It is the most parsimonious approach for diploidized ancient tetraploids
that exhibit gene duplications (Inoue and Kawahara 1990). (3) Treat the plants as if they were diploid and
score each set of homoeologous loci as one locus. This approach is commonly employed for autopolyploid
taxa exhibiting multiple chromosome numbers and polysomic inheritance (Bayer 1989, Cai et al. 1990,
Ehrendorfer et al. 1996, Hamrick and Allard 1972, McArthur et al. 1986). Although most articles providing
statistical analysis of isozyme genetic diversity in polyploids use one of these three approaches, other
methods have been used (Petersen et al. 1993, Sun 1996).
Isozymes provided some evidence that Perideridia erythrorhiza might be tetraploid. First, two invariant
UGPP bands not used in this analysis could be interpreted as a pair of homeologous loci exhibiting a pattern
of fixed heterozygosity. This might suggest either gene duplication in a diploid or a lack of gene silencing in
a tetraploid plant. Second, the PGM-1 locus exhibited variation in staining intensity consistent with the
hypothesis that P. erythrorhiza is autotetraploid. We scored PGM-1 as one locus, ignoring the differences in
staining intensity, but we suspect that the excess heterozygosity observed at this locus results from it being a
pair of homeologous loci. Finally, excess heterozygosity observed at the GOT locus might also be attributed
to the presence of homeologous loci. However, other than the monomorphic bands in UGPP, no fixed
heterozygosity was noted at any locus used in this analysis
Because the chromosome number (38; base number in the complex is 9, 10, or 11) of Perideridia
erythrorhiza is not divisible by four, some loss, amalgamation, or duplication of chromosomes has
obviously occurred. Although all loci of a tetraploid should theoretically contain four alleles, observed band
patterns were not consistent with genetic expectations (perhaps due to gene silencing or aneuploidy). We
therefore treated the plants as if they were diploid and scored each set of homoeologous loci as one locus.
Fourteen loci were scored, representing forms of eleven enzymes. All scored loci were polymorphic. What
were probably two additional loci resolved in UGPP-2, and were invariant in all populations. Because the
focus of this study was taxonomic, they were not included in analysis.
Genetic Variation. Isozyme variation was extremely high in Perideridia erythrorhiza at the species and
population levels (Table 1). For example, the lowest percent polymorphic loci observed in a P. erythrorhiza
population (P=50.0) was much higher than the percent polymorphic loci seen in the average plant species
(34%; Hamrick and Godt 1990). This level of within-population variation would be unusual in a common,
widespread species and is unexpected in such a rare taxon. The most variable population of P. erythrorhiza
(not including the Leather site) is the Illinois River population. The other populations contain comparable
levels of diversity. The Illinois River-bulk population contains very high levels of diversity (data not
shown). Expected heterozygosity is 0.273, observed heterozygosity equals 0.173, percent polymorphic loci
is 100%, and mean number of alleles per locus equals 2.5. The higher diversity levels observed in the bulked
population may be at least a partial result of (1) high levels of within-family variation, and/or (2) sampling
of the bulk lot over a larger geographic area compared to the other Illinois River collection.
Compared to the Mud Flat site, the Pelican Barn population contains slightly less alleles per locus (1.5 vs
1.9) and polymorphic loci (50.0 vs 57.1) (Table 1). However, Pelican Barn contains greater expected
heterozygosity (0.169 vs 0.159). The low observed heterozygosity and high F value in Pelican Barn may be
indicitive of some inbreeding or selfing occurring in the population.
Wright's F statistics were high for most populations and for the species as a whole (Table 1). High
within-population values for F often result from inbreeding, self-pollination, or unrecognized subdivisions
within the population.
T1
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Taxonomy. Isozyme variation does indicate that populations and Regions are highly differentiated,
which is consistent with morphological, phenological, and physiological data (Meinke 1998). The isozyme
data do not support dividing Perideridia erythrorhiza into two groups, east and west of the Cascade crest.
Some populations of P. erythrorhiza had unique alleles, and some alleles were unique to regions.
The Leather population submitted as Perideridia erythrorhiza is probably not that species. It is highly
genetically differentiated from the other populations (Fig. 2; Table 2). Usually, though not always, different
species show fixed (completely consistent) differences at one or more isozyme loci. The distribution of
alleles supported the hypothesis that all samples except Leather and P. oregana were samples from the one
species. All Leather samples had the genotype '44' at UGPP-2; no other sample had the UGPP-2 '4' allele. In
addition, the Leather samples consistently had faint, unscorable bands at MDH-2. Compare this with the P.
oregana sample, which differed from all other samples because it had the '66' genotype at PGM-1; no other
sample had the PGM-1 '6' allele. In addition, the P. oregana samples consistently had faint, unscorable
bands at FEST-1. The differences between the Leather Site sample and P. erythrorhiza, and between the P.
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oregana sample and P. erythrorhiza seem parallel, and the Leather sample is more genetically distinct from
the P. erythrorhiza samples than is P. oregana (Table 2).
It is not possible with the isozyme data to taxonomically identify the Leather population. Perhaps it is a
particularly odd population of Perideridia erythrorhiza, worthy of separate species status. Possibly it is P.
oregana, although isozymes suggest that this is not the case. Perideridia erythrorhiza resembles P.
gairdneri and P. lemmonii; could the Leather population be one of those species? Is it an unrecognized
species? Without voucher specimens or seed to grow out, the identity of this population remains a mystery.
This could be an interesting subject for further research.
F2
T2
Regional Differentiation. As might be expected with such geographically isolated populations, the
genetic identities among some Perideridia erythrorhiza populations were fairly low (excluding the Leather
site) (Table 2). Genetic identities among P. erythrorhiza populations varied from 0.753 to 0.997. Genetic
identities between the P. oregana sample and P. erythrorhiza populations varied from 0.654 to 0.812.
Genetic identities between the Leather Site sample and P. erythrorhiza populations varied from 0.442 to
0.552, and its identity with the P. oregana sample was 0.410.
When these genetic identities are diagramed, the Perideridia erythrorhiza populations cluster together as
would be expected geographically (Fig. 2). The three Klamath Falls populations form one cluster, the two
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Roseburg populations form another, and the Illinois River population is sister group to this pair of clusters.
Note that this dendrogram does not support division of P. erythrorhiza into just two groups, the eastern
Klamath Falls and a western group including the Illinois Valley, Roseburg, and Umpqua populations.
As expected, the one P. oregana sample is distant from all P. erythrorhiza. The Leather population is
more distant still, which would be highly unusual if it were P. erythrorhiza, but, as discussed previously,
presumably it is not.
Wright's F-statistics provide another way to examine the pattern of variation among populations.
Perideridia erythrorhiza populations are highly differentiated (Fpt = 0.388). It has been assumed that
variation in P. erythrorhiza can best be divided into two groups, an eastern group (including populations
Howard Prairie, Mud Flat, and Pelican Barn) and a western group (including populations Illinois River,
Roseburg, and Umpqua). We did not find that division to best express the pattern of isozyme variation we
observed. In a hierarchy of populations within east/west groups, populations are highly differentiated (Fpg =
0.422) and the east/west grouping does not contribute to the pattern of differentiation (Fgt = -0.058, which is
approximately zero; the negative number is an error in the computer program).
A more useful way to express the pattern of variation is to divide the populations into three regions;
Roseburg (the Roseburg and Umpqua populations), Cave Junction (the Illinois River population), and
Klamath Falls (the Howard Prairie, Mud Flat, and Pelican Barn populations). Population differentiation is
somewhat lower though still great (Fpr = 0.274) and regional differentiation is moderate (Frt = 0.157). Of
course, this system should be tested using at least two populations in the Cave Junction region.
Genetic identities support this division of Perideridia erythrorhiza populations into three regions, rather
than two. When the populations are divided into eastern and western groups, the genetic similarity of the
eastern and western group is only slightly lower than the genetic similarity of the western populations
among themselves. In fact, when the samples are divided into three regions, the Klamath and Roseburg
regions are more similar to each other (0.829) than either is to the Cave Junction region (0.789 and 0.805,
respectively).
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ACKNOWLEDGEMENTS
Project cooperator Paul Berrang developed the study proposal, carried out seed collection, germinated the
seed, and transported the seedlings to NFGEL. Laboratory technical support was provided by Suellen
Carroll, Randy Meyer, Pat Guge, and Robert Saich. Funding was provided by the USDA Forest Service,
Region 6 and the Bureau of Land Management. This is a report of the findings of the National Forest
Genetic Electrophoresis Laboratory's Project 78.
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Genetic Differentiation In A Rare Plant With A Disjunct Range: Lewisia
Kelloggii
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ABSTRACT
Lewisia kelloggii isozyme variation was studied in six Idaho and seven California
populations. Lewisia kelloggii is polyploid, and was treated in analysis as an allotetraploid. The
species is highly variable genetically (85% of loci polymorphic, 3.3 alleles per locus) and
populations are highly differentiated (Fst = 0.77). All California and the Greencreek Lake
populations are diverse, but most sampled Idaho populations have relatively little
within-population variation. Isozyme data support the hypothesis that the Idaho and California
populations may be two different, though related, species. For example, genetic identities
between the Idaho and California populations (0.47 - 0.70) are typical of cogeneric species
rather than conspecific populations.
INTRODUCTION
Lewisia kelloggii K. Brandegee is a small rare plant found in open areas on excessively drained,
coarse-textured granitic and volcanic soils on ridgelines. It has a disjunct range, occurring in the Sierra
Nevada of California (from Plumas County south to Fresno County) and in Idaho (in Valley, Elmore, and
Custer Counties).
Lewisia kelloggii has no special seed dispersal mechanisms that would allow dispersal across the 540
km separating the closest California and Idaho populations. The capsule splits open around the sides so that
the top falls off, sometimes with a few seeds adhering. The small (2 mm) round seeds have no special
mechanisms for dispersal. They may be shaken or blown out of the capsule (Matthew 1989), but often
flowers are buried in blowing sand before seed maturation. In these cases, rodents digging to eat the fleshy
taproot are important dispersal agents (Davidson 2000).
Its high elevation habitat might seem to protect this rare species from human disturbance, but that is not
entirely true. Lewisia kelloggii is at risk in part because its open ridgetop habitats are often desirable sites for
roads and for trails. It is vulnerable to damage by logging because its habitats are suitable for landings and
for parking equipment. Lewisia kelloggii is probably tolerant of wildfire due to its geophytic growth form
and open habitat, but it is vulnerable to damage during firefighting operations, when its ridgeline habitats
may be preferred sites for establishing fire control lines and the plants may be killed by bulldozing or soil
compaction. In addition, a few populations have suffered from overcollection for cultivation (K. van Zuuk,
Forest Botanist, Tahoe National Forest, pers. comm.), although the diminutive, nearly stemless species is not
as showy as most Lewisias and is difficult to maintain in cultivation (Matthew 1989).
The taxonomic history of Lewisia kelloggii has been relatively simple (Table 1). Lewisia kelloggii was
published based on a specimen from Placer County, California (Brandegee 1894). Although it has been
stated that the type specimen was lost in the fire that followed the San Francisco earthquake of 1906
(Davidson 2000), it is still at the California Academy of Sciences (B. Bartholomew, pers. comm.).
Subsequently the plant was incorporated into another species (as L. rediviva var. yosemitana; Hall and Hall
1912) but that move was clearly an error, both because L. kelloggii differs in important ways from L.
rediviva, and because the name L. rediviva var. yosemitana K. Brandegee is a synonym of a different
small-statured species now called L. disepala Rydb. Later, the California species L. yosemitana Jeps. was
described within L. kelloggii based on Jepson 4357, 6 July 1911, Summit of El Capitan, Yosemite (B. Ertter,
pers. comm). Lewisia yosemitana was distinguished from L. kelloggii because L. yosemitana had 16 - 26
stamens; L. kelloggii had 10 - 15 stamens. That distinction did not win wide acceptance, and L. kelloggii as
currently understood has a highly variable stamen number. The type specimen of L. yosemitana has been
treated as the neotype of L. kelloggii (Davidson 2000), but that is unnecessary because the holotype still
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exists. Recently, L. kelloggii ssp. hutchisonii L. T. Dempster was described from a single specimen collected
on Saddleback Mountain in Sierra County, in the northern part of the species' California range (Dempster
1996). This subspecies is much larger than typical L. kelloggii; L. kelloggii ssp. hutchisonii has leaves 4 - 10
cm long and pteals 25 - 30 mm long, while L. kelloggii ssp. kelloggii has leaves 2 - 6 cm long and petals 10
mm long. To summarize, at this time the plants in Idaho and California are treated as one species, L.
kelloggii. The only intraspecific taxon recognized (L. kelloggii ssp. hutchisonii), may have a very limited
range in the northern Sierra Nevada.
Recently, small morphological differences between the Idaho and California populations have raised the
possibility that they should be considered two separate species (D. Taylor, pers. comm. to T. Prendusi,
Regional Botanist, Region 4, USDA Forest Service). This would have implications for Lewisia kelloggii
nomenclature, conservation, and legal status. These concerns prompted this study of L. kelloggii genetic
diversity using isozymes. T1
METHODS
Two to three leaves per plant were collected from each of approximately 30 plants per population in
seven California and six Idaho populations (Table 2). Three pairs of populations were collected very close
together. The Burnt Creek and Noname Creek populations were within 0.6 mile on Red Mountain, Idaho.
The two Soda Springs populations were collected within 2 miles. The two populations from the Plumas
National Forest were a few hundred feet apart.
For further details of isozyme electrophoresis and data analysis, see Appendix 1.
T2
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RESULTS
Lewisia kelloggii was obviously polyploid. For evidence, see
Appendix 2.
Lewisia kelloggii was highly variable. Overall, 85% of the loci were polymorphic, with more than three
alleles per locus (Table 3). In the California populations, 39% to 58% of the loci were polymorphic, but in
Idaho, five of the six populations had less than 20% polymorphic loci.
Populations were highly differentiated genetically, with Fst of 0.40 or higher in each state and the study
as a whole (Table 4). About half the genetic differentiation in L. kelloggii was variation between states
(Table 4). Inferred gene flow among populations was therefore very low, especially within Idaho and
between Idaho and California (Table 3). Each state had unique alleles. Eight of the unique California alleles
and three of the unique Idaho alleles occurred at frequencies greater than 0.80. There were other consistent
isozyme differences between the states.
Genetic identities among populations were highly variable (Table 5). When these genetic identities are
expressed diagrammatically, the populations fall easily into two groups, one from California and one from
Idaho (Figure 1). The mean geneic idenity between California and Idaho populations is 0.58 Table 6),
extremely low for conspecific populations (Crawford 1989).
dendrogram
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Within states, about half the variation was within populations, and half was differentiation among
populations (Table 4). Four California and three Idaho populations had unique alleles. Within California,
populations on the same National Forest clustered together (Figure 1). Genetic identity among California
populations varied from 0.69 to 0.99, and within Idaho varied from 0.76 to 0.99. Populations within 2 miles
of each other were particularly similar (0.9649 for Plumas A and B, 0.9978 for Soda Springs 1 & 2, and
0.9916 for Burnt Creek and Noname Creek; Table 5).
Two Idaho populations had particularly interesting patterns of isozyme diversity. Whitehawk Summit
population had no unique alleles, but every individual had certain alleles (PGI2 - 5, PGM2 - 3, and UGPP2 2) otherwise rare in Idaho and therefore its genetic identities with other Idaho populations were relatively
low (averaging 0.82; Table 6). The Greencreek Lake population was by far the most diverse in Idaho, with
48% polymorphic loci (compared to less than 20% for each of the other Idaho populations; Tables 3 and 5).
The California population from Shuteye Peak was particularly distinct. Not only were genetic identities
between the Shuteye Peak population and other California populations low (averaging 0.73; Table 6), but
the population had four unique alleles and had other alleles (e.g. MDH2 allele 2) found elsewhere only in
Idaho.
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DISCUSSION
Genetic interpretation. Polyploidy complicated the analysis of Lewisia kelloggii isozyme variation. For
details, see Appendix 2.
Taxonomy and genetic variation. As a species, Lewisia kelloggii showed great isozyme variation.
Measures of isozyme diversity were much higher than average for plants with similar characteristics, and
high even for widespread species (Hamrick and Godt 1990). Within each state, the variation was about as
great as expected of widespread, insect-pollinated, herbaceous species. Of course, L. kelloggii is not
widespread.
Within California, the Shuteye Peak population (Sierra National Forest) was particularly divergent. It
was the southernmost population sampled in this study, and may represent a different subspecies or variety
than the more northern populations. Genetic identities between Shuteye Peak and more northern California
populations averaged 0.71 (range 0.66 to 0.74; Table 6), compared to expected identities of 0.90 or greater
for members of the same species and subspecies. The Shuteye Peak population had some genes and band
patterns that were unusual for California but were shared with the Idaho population (including the MDH1
pattern, and allele 2 of MDH2). Although earlier attempts to split off southern L. kelloggii as a separate
taxon from the northern plants were unsuccessful (Hall and Hall 1912, Jepson 1923), perhaps the issue
should be revisited.
Interestingly, for certain enzymes (e.g. MDH), some individuals in the two Eldorado National Forest
have alleles that are typical of the northern populations and other individuals have the alleles seen in the
southern Shuteye Peak population.
Lewisia kelloggii ssp. hutchisonii was recently described from northern California (Dempster 1996). The
two populations sampled from the Plumas National Forest (Table 2) were identified as L. kelloggii ssp.
hutchisonii (L. Janeway 1998, unpublished report to Plumas National Forest). In the absence of voucher
specimens, we can not confirm that identification. If it is correct, and if the other northern specimens were L.
kelloggii ssp. kelloggii, then L. kelloggii ssp. hutchisonii is so genetically similar to L. kelloggii ssp.
kelloggii that the usefulness of this name is questionable. Alternatively, the name L. kelloggii ssp.
hutchisonii may have been applied more broadly to these populations than is appropriate.
Three types of isozyme data show that the Idaho and California populations of Lewisia kelloggii probably
should be considered two different species. First, about half the isozyme variation in the entire study was
variation between the states (Table 4). Second, the genetic identities between Idaho and California
populations averaged 0.58 (Table 6). In general, plant populations within the same species (and subspecies)
have genetic identities greater than 0.90, and populations of different congeneric species have genetic
identities averaging 0.68, though varying from 0.25 to 1.00 (Crawford 1989). Third, there were consistent
differences between Idaho and California populations in several isozymes (Tables 5, 6 and 7). Consistent
differences in isozymes are characteristic of pairs of closely related plant species. Obviously, the Idaho and
California populations do not share the same gene pool. They are genetically isolated and have been isolated
for a long time.
Outcrossing plants that are as genetically divergent as the Idaho and California populations of Lewisia
kelloggii are usually treated as separate species. We hesitate to make such a recommendation based on
isozyme variation alone, simply because species that have to be identified by electrophoresis have limited
practical use. However, we strongly recommend that the present study be followed up with a morphological
analysis of Lewisia kelloggii. If the recent suggestion that there are morphological differences between the
plants growing in the two states
(D. Taylor, pers. comm. to T. Prendusi, Regional Botanist, Region 4, USDA Forest Service) is confirmed,
the Idaho populations should be formally described as a separate species.
Conservation. Lewisia kelloggii populations in Idaho and California should be managed as distinct
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entities. They are as distinct as two different species and perhaps should be treated taxonomically as species.
Both of these genetically distinct units should be preserved.
Conserving the great isozyme variation seen in Lewisia kelloggii requires preserving the plants in each
region (e.g. each National Forest) where it occurs. Individual populations are highly differentiated; about
half the variation with states was variation among populations. However, groups of populations growing
within two miles of each other can be managed as units because they are very similar.
In Idaho, two populations should be particularly high priorities for conservation. Most Idaho populations
showed relatively little variation, but the Greencreek Lake population was as variable as California
populations (Table 3). It had genes not observed in the other sampled Idaho populations. Some of these
appear to be genes shared with California populations. The Whitehawk Summit population had very little
variation, but some of its genes were unusual for Idaho, and it was the most genetically divergent of that
state's populations.
In California, the National Forests were all differentiated, suggesting that it is important to conserve this
rare plant in every National Forest where it occurs. The Shuteye Peak population (Sierra National Forest)
was particularly divergent. We do not know if it is typical of southern populations because it was the only
southern population sampled. At least until more is known about genetic diversity in southern L. kelloggii,
the distinctive Shuteye Peak population should be a priority for conservation.
ACKNOWLEDGEMENTS
We thank Suellen Carroll, Patricia Guge, and Randy Meyer at the National Forest Genetic
Electrophoresis Laboratory for technical support. We thank Bruce Bartholemew at CAS and Barbara Ertter
at UC for information about Lewisia kelloggii type specimens. The project was initiated by Chris Frisbee,
USDA Forest Service, and is NFGEL Project #74. This report was prepared by Barbara Wilson and Valerie
Hipkins.
REFERENCES
Brandegee, K. 1894. Studies in Portulacaceae. Proc. Calif. Acad. Sci.
Series 2, 4: 86-91.
Conkle, M. T., Hodgskiss, P. D., Nunnally, L. B., and Hunter, S. C. 1982.
Starch Gel Electrophoresis of Conifer Seeds: a Laboratory Manual. Gen. Tech. Rep. PSW-64.
Berkeley, CA: Pacific Southwest Forest and Range Experiment Station, Forest Service, U.S.
Department of Agriculture. 18pp.
Crawford, D. J. 1989. Enzyme electrophoresis and plant systematics. pp.
146 - 164 in D. E. & P. S. Soltis, eds. Isozymes in Plant Biology. Dioscorides Press, Portland,
Oregon.
Curran, in 1894. Brandegee, K. 1894. Studies in Portulacaceae. Proc.
Calif. Acad. Sci. Series 2, 4: 86-92.
Davidson, B. L. 2000. Lewisias. Timber Press, Portland, Oregon.
Dempster, L. T. 1996. A new subspecies of Lewisia (Portulacaceae) in
California Madroño 43: 415-416.
Gottlieb, L. D. 1981. Electrophoretic evidence and plant populations.
Prog. Phytochem. 7: 1-46.
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Gottlieb, L. D. 1982. Conservation and duplication of isozymes in plants.
Science 216: 373-380.
Hall, H. M. and C. C. Hall. 1912. A Yosemite Flora. Paul Elder & Co.,
San Francisco.
Hamrick, J. L., and Godt, M. J. W. 1990. Allozyme diversity in plant
species, pp. 43-63 in Brown, A. H. D., Clegg, M. T., Kahler, A. L., and Weir, B. S., eds., Plant
Population Genetics, Breeding, and Genetic Resources. Sunderland, Massachusetts: Sinauer
Associates
Hartl, D. L, and Clark, A. G. 1989. Principles of Population Genetics, 2nd
edition. Sinauer Associates, Sunderland, Massachusetts.
Hershkovitz, Mark A. 1990. Nomenclatural changes in Portulacaceae.
Phytologia 68: 267-270.
Jepson, W. L. 1923. A Manual of the Flowering Plants of California.
University of California, Berkeley, California. (p. 352)
Kimura, M. and J. F. Crow. 1964. The number of alleles that can be
maintained in a finite population. Genetics 49: 725-738.
Lewontin, R. C. 1972. The apportionment of human diversity.
Evolutionary Biology 6: 381 - 398.
Matthew, B. 1989. The Genus Lewisia. Timber Press, Portland, Oregon.
Missouri Botanic Garden. (website accessed 30 August 2001). Index to
Plant Chromosome Numbers (IPCN). http://mobot.mobot.org./W3T/search/ipcn.htm.
Moldenke, A. R. 1973. A contribution to a chormosome atlas of the
California flora. Technial Report 73-22. University of Califoria, Santa Cruz, California.
Nei, M. 1973. Analysis of gene diversity in subdivided Populations. Proc.
Nat. Acad. Sci. USA 70(12): 3321-3323
Nei, M. 1978. Estimation of average heterozygosity and genetic distance
from a small number of individuals. Genetics 89: 583-590.
Pitel, J. A., and W. M. Cheliak. 1984. Effects of extraction buffers . . . .
Rydberg. 1932. North American Flora 21: 326.
Soltis, D. E., and Rieseberg, L. H. 1986. Autopolyploidy in Tolmiea
menziesii (Saxifragaceae): genetic insights from enzymes electrophoresis. American Journal of
Botany 73: 310-318.
USDA Forest Service. 2000. National Forest Genetic Electrophoresis
Laboratory Standard Operating Procedures. NFGEL, USDA Forest Service, Camino, California.
Weeden, N. F. and Wendel., J. F. 1989. Genetic of plant isozymes. pp. 4672 in Isozymes in Plant Biology, eds. D. E. Soltis and P. S. Soltis. Dioscorides Press, Portland,
Oregon.
Wefald, M. (accessed August 2001) TRS2LL. Online. Available
http://www.crl.com/~wefald/trs2ll.html.
Weir, B. S. 1990. Genetic Data Analysis. Sunderland, Massachusetts:
Sinauer.
Yeh, F. C., Yang, R.-C., and Boyle, T. 1997. Popgene Version 1.20.
University of Alberta, Edmonton, Alberta.
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T3
T4
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Appendix1
APPENDIX 1: METHODS
Two to three leaves per plant were collected from each of approximately 30 plants per population in
seven California and six Idaho populations (Table 1). Leaves from each individual were bagged separately
from those of other individuals, and they were shipped to NFGEL on ice. Collection locations were reported
in terms of TRS and converted to latitude/longitude using the program TRS2ll (Wefald 2001). One 7 mm
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diameter leaf disk was submerged in each of three microtiter plate wells containing two drops (100 µl) of a
0.1 M Tris-HCl (pH 8.0) extraction buffer, with 10% (w/v) polyvinylpyrrolidone-40, 10% sucrose, 0.17%
EDTA (Na2 salt), 0.15% dithiothreitol, 0.02% ascorbic acid, 0.10% bovine albumin, 0.05% NAD, 0.035%
NADP, and 0.005% pyridoxal-5-phosphate (UDSA Forest Service 2000; Pitel & Cheliak 1984). Samples
were frozen at -70ºC. On the day of electrophoresis, samples were thawed and ground and the extracts were
absorbed onto 3 mm wide wicks prepared from Whatman 3MM chromatography paper.
Methods of sample preparation and electrophoresis follow the general methodology of Conkle et al.
(1982), with some modifications (USDA Forest Service 2000). All enzymes were resolved on 11% starch
gels. A lithium borate electrode buffer (pH 8.3) was used with a Tris citrate gel buffer (pH 8.3) (Conkle et
al. 1982) to resolve alcohol dehydrogenase (ADH), fluorescent esterase (FEST), leucine aminopeptidase
(LAP), phosphoglucomutase (PGM), and phosphoglucose isomerase (PGI). A sodium borate electrode
buffer (pH 8.0) was used with a Tris citrate gel buffer (pH 8.8) (Conkle et al. 1982) to resolve
glucose-6-phosphate dehydrogenase (G6PDH), glutamate-oxaloacetate transaminase (GOT),
glucose-6-phosphate dehydrogenase (G6PDH), triosephosphate isomerase (TPI), and uridine
diphosphoglucose pyrophosphorylase (UGPP). A morpholine citrate electrode and gel buffer (pH 6.1)
(USDA Forest Service 2000) was used to resolve isocitrate dehydrogenase (IDH), phosphogluconate
dehydrogenase (6PGD), and malate dehydrogenase (MDH). Enzyme stain recipes follow USDA Forest
Service (2000).
Initially, two people independently scored each gel, assuming that Lewisia kelloggii was diploid, like
species examined in a previous study (NFGEL project 36; Lewisia cantelovii, L. condonii, and L. serrata).
When they disagreed, a third person resolved the conflict. For further quality control, 10% of the individuals
were run and scored twice. In addition, gels were photographed for future reference. Genetic interpretations
were inferred directly from isozyme phenotypes based on knowledge of the generally conserved enzyme
substructure, compartmentalization, and isozyme number in higher plants (Gottlieb 1981, 1982; Weeden and
Wendel 1989), and following a previous study of Lewisia isozymes (NFGEL project 36). However, complex
patterns consisting of four or more bands demonstrated that Lewisia kelloggii must be polyploid. The
samples were rescored as tetraploids by one of us (BLW), using photographs of the gels plus the initial
scores, and at least some of the samples in eleven of the populations were run and scored again.
We are not aware of any published chromosome count for Lewisia kelloggii. The data were analyzed as
33 diploid loci. One locus each was scored for ADH, FEST, and PGM1. Two loci were scored for G6PDH,
GOT2, IDH1, LAP2, MDH2, PGI2, PGM2, TPI1, TPI2, UGPP1, UGPP2, 6PGD1, and 6PGD2. Four loci
were scored for MDH1. (See Discussion for explanation). For enzymes scored as pairs of loci, an isozyme
band pattern consisting of a single band was considered to consist of two homeologous loci. All alleles
detected in this study were assigned to only one of the two homoeologous loci, unless the observed band
combinations or intensities suggested that two alleles belonged to different loci. That is, we assumed one
locus of each pair of homeologous loci was invariant, unless band patterns indicated this was not the case. In
addition, one haploid locus was scored for PGI1 (which is encoded in the chloroplast) but it was not
included in analysis.
Results were analyzed using Popgene, version 1.21 (Yeh et al. 1997). A locus was considered
polymorphic if an alternate allele occurred even once. We calculated unbiased genetic distances (Nei 1978),
expected heterozygosity (Nei 1973), and gene flow [Nm (the effective number of migrants per year) =
0.25(1-Fst)/Fst; Slatkin and Barton 1989]. The fixation indices for populations (F) were calculated in
Popgene (Yeh et al. 1997) following Hartl and Clark (1989), but F statistics for the hierarchy of regions
within the species (Fpt), populations within the species (Fst), populations within regions (Fsp), and
individuals within the species (Fit), regions (Fip), and populations (Fis) were calculated by the method of
Weir (1990); the two methods produced slightly different values for F. Dendrograms based on unbiased
genetic distances (Nei 1978) were generated using UPGMA.
Appendix2
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APPENDIX 2: POLYPLOIDY
Interpreting isozyme band patterns as genotypes requires some knowledge of the plant's diploid or
polyploid nature. We are not aware of a published chromosome number for Lewisia kelloggii. Certainly
none is listed in Moldenke (1973), Matthew (1989) or in the Index of Plant Chromosome Numbers
(Missouri Botanic Garden 2001). This lack of information may result from technical difficulties. Root tip
squashes are unclear in Lewisia, making accurate counts difficult (Matthew 1989). Anthers from very
immature flower buds are preferred, but collecting young enough buds is difficult in a short-stemmed plant
like L. kelloggii even if plants are readily available, which they are not; Lewisia kelloggii is very difficult to
maintain in cultivation (Matthew 1989).
We began our interpretation with the assumption that Lewisia kelloggii is diploid, an assumption that
proved feasible in an earlier study of other Lewisia species (NFGEL project 36, unpublished; Lewisia
cantelovii, L. condonii, and L. serrata). However, isozymes demonstrated that L. kelloggii must be
polyploid. Evidence for the polyploid nature of L. kelloggii includes complicated band patterns with four or
more bands and populations showing fixed or nearly fixed heterozygosity. We assumed that the plant was
tetraploid and scored two loci (four alleles) for each enzyme (each region of activity on the gel). However, a
tetraploid interpretation seemed inappropriate for ADH, FEST, and PGM1 because in some populations
most individuals were homozygous for one of two alleles, heterozygotes were rare, and the heterozygotes all
appeared balanced. Therefore, these enzymes were scored as diploid. The isozyme patterns in MDH1
involved 5 to 6 bands in some populations and could only be explained by the presence of four interacting
loci.
Inferring variable numbers of loci for regions of activity on isozyme gels is unsatisfying, but may be
consistent with chromosome evolution in Lewisia. Published chromosome numbers in the genus include 20,
24, 28, 30, 56, and 66 (Table 7). (The species considered most closely related to L. kelloggii is L.
brachycalyx Engelmann ex A. Gray with 2n = 20.) Apparently Lewisia evolution has featured the loss,
duplication, or fusion of chromosomes, as well as simple polyploidy.
Although L. kelloggii is clearly polyploid, we are not certain that it is tetraploid. Evidence for possible
higher polyploidy included consistent uneven band staining with one homodimer much darker than either
the heterodimer or the other homodimer (GOT, MDH1, PGI2, and 6PGD1) and certain unexpectedly
common multibanded genotypes, such as the one scored as 1136 in PGI2. However, this evidence was
unclear, and we chose to treat L. kelloggii was tetraploid.
We are not certain that the Idaho and California populations have the same chromosome number. The
kind of isozyme band pattern that result from three or more alleles occurred in only one Idaho population,
and fixed heterozygosity was less common in Idaho than in California. These data suggest that Idaho plants
may have a lower chromosome number than California plants, although they may be an artifact of the lower
genetic variation in Idaho populations (Table 3).
Evidence about the mode of inheritance in homeologous loci was mixed. Some enzymes in some
populations exhibited fixed or nearly fixed heterozygosity expected of allotetraploids with disomic
inheritance. On particularly clear gels, such as those for TPI2, unequal staining intensities suggested that the
allele combinations 11 11, 11 14, and 11 44 occurred. If inheritance were autotetraploid or allotetraploid but
tetrasomic, the combinations 1444 and 4444 would have been observed, but they were not. On the other
hand, some enzymes in some populations gave evidence for tetrasomic inheritance (Soltis and Rieseberg
1986). For example, individuals in some populations showed alternate homozygous conditions. In three
cases (ADH, FEST, and PGM1), the alternate homozygous states plus balanced staining in heterozygotes
led us to interpret the enzymes as diploid, but in the other cases we scored the enzyme as tetraploid because
of unequal staining intensities in heterozygotes, fixed heterozygosity in some populations, and/or the
occurrence of some individuals with more bands than could be explained in a diploid. We hypothesized that
L. kelloggii was an allotetraploid with disomic inheritance.
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In order to perform a genetic analysis of Lewisia kelloggii isozymes, we had to make assumptions about
polyploidy and mode of inheritance. Although each assumption is a reasonable hypothesis based on
evidence, it may be wrong; L. kelloggii may not be a tetraploid with disomic inheritance. Fortunately, this
study's implications for taxonomy and conservation genetics are clear and are robust to errors in our
hypotheses.
T7
Table 7. Published chromosome numbers of Lewisia. See Matthew (1989) for references. * = recently moved to a different
genus: Cistanthe tweedyi (A. Gray) Hershkovitz 1990.
Chromosome number
2n = 20
2n = about 24
2n = 28
2n = 30
2n = about 56
2n = about 66
2n = 92
Species
L.brachycalyx G. Engelmann ex A. Gray
L. congdonii (Rydberg) S. Clay
L. cantelovii J. T. Howell
L. cotyledon (S. Watson) B. L. Robinson
L. leeana (T. C. Porter) B. L. Robinson
L. rediviva Pursh
L. columbiana(T. J. Howell ex A. Gray) B. L. Robinson
L. nevadensis (A. Gray) B. L. Robinson
L. pygmaea (A. Gray) B. L. Robinson
L. tweedyi (A. Gray) B. L. Robinson*
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Genetic diversity in Broadleaf Lupine (Lupinus latifolius) accessions from
the Mt. Hood National Forest
MANAGEMENT SUMMARY
OBJECTIVES
1. Estimate levels of genetic diversity within and among populations of broadleaf
lupine. Information from this will be used to answer questions about sampling wild
populations for seed collections. Do we need to sample just a few or many different
populations to provide a broad genetic base for this species?
Presumed diploid and presumed tetraploid populations of Lupinus latifolius all are
highly variable. Individuals are differentiated, but (within each ploidy level)
populations are little differentiated. Therefore, collecting seed from many individuals
from relatively few populations would be sufficient to provide a broad genetic basis
for a cultivated seed source.
2. Verify there are no consistent major differences which might imply different taxa or
hybrids.
Isozyme data do not readily support the hypothesis that the study includes more than
one species. Within each ploidy level, populations are little differentiated. The
presumed tetraploid populations (large populations 1, 7, and 8, and small sample 18)
are strongly differentiated from the remaining populations (although genetic distances
between these populations and the presumed diploid populations are within the range
expected of conspecific plants. Populations 1, 7, 8, 18, and 19 differ from other
populations in allele frequencies, not because they have alternate alleles (fixed
differences). All alleles that were unique to the presumed diploid or presumed
tetraploid populations were rare. Polyploidy and its immediate consequences do not
imply that the presumed tetraploid populations should be recognized taxonomically.
If observed isozyme differentiation coincides with morphological differentiation,
some sort of formal taxonomic separation may be warrented, most likely at an
intraspecific level.
INTRODUCTION
Guidelines for transferring seed of Broadleaf Lupine (Lupinus latifolius J. Agardh; Fabaceae) in
restoration projects will be based in part on its genetic variation, as assessed using both morphological
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and isozyme markers. In 1995 and 1996, seeds were collected from 83 locations on the Mt. Hood
National Forest, Oregon. These seeds were used to establish common gardens with two replications at
each of two facilities, one in Corvallis, Oregon, and one in Carson, Washington. An analysis of
morphological results has been published (Doede et al.1998).
Seed from a subset of the sampled populations was sent to the National Forest Genetic
Electrophoresis Laboratory (NFGEL) for isozyme analysis. Isozymes were used to evaluate how many
wild populations should be sampled to provide an adequate representation of natural genetic diversity.
Identity of the plants was verified by Caitlin Cray, who found that all samples conformed to the
descriptions in Hitchcock et al. 1961. However, taxa in the genus Lupinus are variable, prone to
hybridization, difficult to identify, and often controversial. Therefore, isozymes were also used to verify
that all samples were from one taxon.
METHODS
Sample collection. Seed was collected in the Mt. Hood National Forest in 1995 and 1996. Seeds
collected from each maternal plant were kept separate. One to five seeds were collected from each of 4
to 32 individuals per population. Seed submitted for this study came from a total of 339 individuals from
19 populations (Table 1).
Sample Preparation. One seed from each parent plant was nicked with a scalpel and soaked in
water for two hours. The seed coat was then removed and each seed was placed in a microtiter plate well
containing 150 µl of a Tris buffer (pH 7.5; Gottlieb 1981). The plates were frozen at -70°C. On the
morning of electrophoresis, the samples were thawed and ground, and the slurry was absorbed onto five
3-mm wide wicks prepared from Whatman 3MM chromatography paper. Two of the five wicks were
returned to the freezer to serve as backup samples.
Electrophoresis. Methods of electrophoresis are outlined in USDA Forest Service (2000), and
follow the general methodology of Conkle et al. (1982) except that most enzyme stains are modified.
The following enzymes were examined: aconitase (ACO), alcohol dehydrogenase (ADH), acid
phosphatase (ACP), catalase (CAT), fluorescent esterase (FEST), glyceraldehyde-2-dehydrogenase
(GLY), glutamate-oxaloacetate transaminase (GOT), isocitrate dehydrogenase (IDH), leucine
aminopeptidase (LAP), malate dehydrogenase (MDH), malic enzyme (ME), phosphoglucomutase
(PGM), phosphogluconate dehydrogenase (6PGD), phosphoglucose isomerase (PGI), shikimic acid
dehydrogenase (SKD), and triosephosphate isomerase (TPI). All scored enzymes migrated anodally,
except that ACP-2 migrated cathodally. A total of 19 loci resolved sufficiently well to use in genetic
analysis. A lithium borate electrode buffer (pH 8.3) was used with a Tris citrate gel buffer (pH 8.3)
(Conkle et al. 1982) to resolve ACO, ADH, FEST-1, FEST-3, LAP, ME, and PGM. A sodium borate
electrode buffer (pH 8.0) was used with a Tris citrate gel buffer (pH 8.8) (Conkle et al. 1982) to resolve
CAT, GOT-1, GOT-2, GLY, TPI-1, and TPI-2. A morpholine citrate electrode and gel buffer (pH 6.0)
(USDA Forest Service 2000) was used to resolve ACP-2, IDH, 6PGD, and SKD. All enzymes were
resolved on 11% starch gels. Enzyme stain recipes follow USDA Forest Service (2000). Two people
independently scored each gel. When they disagreed, a third person resolved the conflict. For quality
control, 10% of the individuals were run and scored twice.
Data Analysis. Genetic interpretations were inferred directly from isozyme phenotypes. Most
isozyme variants are inherited in a Mendelian fashion in legumes. Therefore, genetic interpretations were
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inferred directly from isozyme phenotypes, based on knowledge of the generally conserved enzyme
substructure, compartmentalization, and isozyme number in higher plants (Gottlieb 1981, 1982; Weeden
and Wendel 1989).
Lupinus latifolius can be diploid or tetraploid (Hitchcock et al. 1961). Populations 1, 7, and 8 were
treated as tetraploid because many individuals in each were unambiguously tetraploid, as determined by
the presence of three-banded patterns in monomeric enzymes or four-banded patterns in dimeric
enzymes. Other populations were treated as diploid. There are no standardized approaches for assessing
genetic diversity in population that includes both diploids and autotetraploids. Three different datasets
wre constructed and used for quantifying genetic diversity. (1) In the 25-locus dataset, GLYDH, GOT2,
LAP1, PGI2, PGM1, and SKD were each represented by two loci each. In tetraploid populations,
individuals scored as having only two alleles were scored as having missing data at the second
homoeologous locus, and in diploid populations the second locus was scored as homozygous for a null
allele. (2) In the 19-locus dataset, GLYDH, GOT2, LAP1, PGI2, PGM1, and SKD were represented by
one locus each. For those individuals with three or four alleles at one of these loci, that locus was scored
as having missing data. (3) In the 13-locus dataset, GLYDH, GOT2, LAP1, PGI2, PGM1, and SKD were
entirely removed.
Results were analyzed using Popgene version 1.21 (Yeh et al. 1997) and Biosys-1, version 1.7
(Swofford and Selander 1989). A locus was considered polymorphic if an alternate allele occurred even
once. Statistics calculated included unbiased genetic distances (Nei 1978), expected heterozygosity
(Levene 1949), expected number of alleles/locus (Kimura and Crow 1970), and gene flow
(Nm = 0.25[1/Fs]/Fst; Slatkin and Barton 1989). A dendrogram was generated in Popgene using
UPGMA and Nei's unbiased genetic distances. Hierarchical F statistics (Wright 1978) were generated
using Biosys-1, version 1.7 (Swofford and Selander 1989), except that F statistics for the three-level
hierarchy (Table 4) were generated using the method of Weir (1990) using Popgene (Yeh et al. 1997).
RESULTS
Unbalanced heterozygous genotypes or evidence of three or four alleles per locus were observed in 37
individuals, most in populations 1, 7, and 8. Unambiguously tetraploid genotypes occurred in six loci,
GLYDH, GOT2, LAP1, PGI2, PGM1, and SKD. The other 13 loci examined could be scored as diploid
in all individuals. For analysis we treated populations 1, 7, and 8 as presumed tetraploids and populations
2, 3, 4, 5, 6, 9, and 10 as presumed diploids. Possible polyploidy could not be evaluated in the small
populations (populations 11 through 19) due to sample size of four plants each, except that one tetraploid
was detected in population 18.
To determine the mode of inheritance, genotype frequencies were calculated for all 19 loci (or sets of
homoeologous loci) in the 37 individuals that were unambiguously tetraploid. Twelve of these loci
provided evidence for tetrasomic inheritance. None had fixed heterozygosity, which would have
provided evidence for disomic inheritance.
In order to generate genetic diversity statistics and the cluster diagrams based on them using software
designed for diploid plants, three datasets were produced. (1) In the 25-locus dataset, GLYDH, GOT2,
LAP1, PGI2, PGM1, and SKD were each represented by two loci each. In tetraploid populations,
individuals scored as having only two alleles were scored as having missing data at the second
homoeologous locus, and in diploid populations the second locus was scored as homozygous for a null
allele. (2) In the 19-locus dataset, GLYDH, GOT2, LAP1, PGI2, PGM1, and SKD were represented by
one locus each. For those individuals with three or four alleles at one of these loci, that locus was scored
as having missing data. (3) In the 13-locus dataset, GLYDH, GOT2, LAP1, PGI2, PGM1, and SKD were
entirely removed. Allele frequencies were calculated from these datasets under the assumption that the
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plants were diploid.
Both diploid and tetraploid populations were variable, with 89% of loci polymorphic loci and more
than three alleles per locus (Tables 1 - 4). Most alleles occurred in both large diploid and large tetraploid
populations. The ten alleles observed in only diploids or tetraploids were rare (frequency <0.05). Five of
these alleles also occurred in small populations.
Genetic identities within a species are usually greater than 0.9 (Crawford 1989). Genetic identities
among the ten large samples of Lupinus latifolius were greater than 0.9 (Tables 5 - 7; summarized in
Table 8), except that genetic identities between tetraploid Population 1 and diploid populations varied
from 0.88 and 0.901 (using the 19 locus dataset; Table 6). Population 19, represented by only four
samples, differed greatly from other populations (Table 6). Within each ploidy level, populations were
little differentiated (Fpt < 0.03; Table 9), but the two ploidy levels were greatly differentiated (Fpt >
0.15; Table 10).
No matter which dataset was used, the similarities among populations followed the same pattern
(Figures 1 - 3). Large presumed diploid populations clustered with each other and with most small
population. The three large presumed tetraploid populations clustered together. Small population 18,
which had one clearly tetraploid individual, clustered more or less near the tetraploids. Small population
19 was divergent due to its unusual combination of allele frequencies, not rare alleles.
DISCUSSION
Polyploidy. Species presenting the most difficult problems for scoring isozymes are those that have
two or more ploidy levels, tetrasomic inheritance, and dosage regulated gene expression. Unfortunately,
Lupinus latifolius is such a species.
Some individuals of Lupinus latifolius are diploid (2n = 2X =24), and others are tetraploid (2n = 4X =
48; Hitchcock et al. 1961). In this study, L. latifolius inheritance appeared tetrasomic; unbalanced
heterozygotes and alternate homozygous states were observed. Polyploids with tetrasomic inheritance
have more possible genotypes than tetraploids with disomic inheritance. Many of these genotypes are
difficult to distinguish, particularly the balanced heterozygotes (AABB) and unbalanced heterozygotes
(AAAB and ABBB).
Dosage regulation of gene expression contributed to scoring difficulty in Lupinus latifolius. If protein
production is dosage regulated, each set of homologous or homoeologous loci will produce the same
total amount of protein, no matter how many loci contribute to that total. On gels, the intensity of a band
is roughly proportional to the amount of protein. Production of the proteins we studied appeared to be
dosage regulated in Lupinus latifolius because the bands produced by homozygous genotypes were
equally dark in diploid and polyploid plants. Each band in a heterozygote is less intense than the single
band of a homozygote, and bands are not equally dark. In a polyploid with three or more alleles at a
locus, some bands are 1/8 to 1/16 as dark as the single band of a homozygote. These faint bands may be
difficult or impossible to see. Our understanding of polyploid isozyme patterns helps us compensate for
this missing information, but scoring gels with faint or missing bands is a slow, difficult process.
Despite these difficulties, NFGEL personnel inferred alleles from the Lupinus latifolius isozyme
patterns and produced a data set of allele occurrences. Unfortunately, no software is available to provide
genetic diversity or genetic distance statistics for tetraploids with tetrasomic inheritance. There is no
consensus on how to deal with taxa which have both diploid and tetraploid individuals or populations.
The most common treatment is to force all scores to a diploid format and use standard genetics software.
We did this with the L. latifolius data set, as detailed in results. We provided separate analyses of
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presumed diploid and tetraploid populations, as well as a combined analysis.
Isozymes patterns of 38 individuals in one small and four large populations exhibited characteristics
of tetraploids; uneven band staining associated with unbalanced heterozygosity or more bands than could
be produced by a diploid. Most individuals and most populations in this study appeared diploid.
However, or any given locus, a tetraploid individual may have an isozyme pattern that could be scored as
diploid. Some tetraploids may have diploid-like patterns at every observed locus. Therefore, we cannot
be sure that each sampled population has a consistent chromosome number.
We chose to treat populations 1, 7, and 8 as tetraploid because eight to fifteen individuals had
isozyme patterns typical of tetraploids, including patterns demonstrating three or more alleles per locus.
We treated all the others as diploid, although three individuals in population 6 had isozyme patterns that
we interpreted as unbalanced heterozygotes.
Lupinus latifolius appears to be an autopolyploid. Its isozyme patterns show that inheritance is
tetrasomic.
We recommend determining chromosome numbers of these Lupinus latifolius individuals before any
further molecular work, whether using DNA or isozymes. Chromosome numbers of individual plants can
be determined efficiently through flow cytometry. Live leaf tissue is assayed. The Seed Lab at Oregon
State University, Corvallis, Oregon, performs this laboratory technique commercially. Normally, a
standard of known chromosome number is used for comparison, but in this case that would not be
necessary, as long as the tested sample included both diploids and tetraploids.
Genetic diversity and population differentiation. Both diploid and tetraploid populations were
highly variable, with 90% polymorphic loci and an average of more than three alleles per locus.
In all populations, observed heterozygosity was substantially lower expected and thus the inbreeding
coefficient F was greater than zero (Tables 1 - 4). The values seen in diploid populations suggestthat
considerable selfing or other inbreeding occurred. Inbreeding coefficients were lower for tetraploid
populations (Tables 1 - 4). That does not imply that they had less inbreeding than the diploid
populations. Inbreeding coefficients are calculated by comparing observed and expected
heterozygosities. The expected heterozygosities used here (Tables 1 - 4) are based on the assumption that
the populations are diploid (Levene 1949). Heterozygosity is expected to be much greater in
autotetraploids than in diploids. The lower inbreeding coefficients for tetraploid Lupinus latifolius
populations show that in tetraploids, heterozygosity was closer to that expected for diploids than it was
in diploids. That is more likely the result of polyploidy itself, rather than breeding system. More
accurately calculating inbreeding rates for autotetraploids is complicated.
When diploid populations were compared to other diploids, or when tetraploids were compared with
tetraploids, population differentiation was low (Fpt = 0.03). However, diploid populations were greatly
differentiated from tetraploids (Fpt = 0.1653).
Taxonomy. Isozyme data do not readily support the hypothesis that more than one taxon was
included in this study. The most divergent population (population 19) had the same alleles as other
populations, differing only in allele frequencies. This population was represented by only four
individuals and therefore deviation from expected allele frequencies may be due to chance.
Larger Lupinus latifolius populations formed two groups, which seemed to correspond with
chromosome numbers. Polyploids usually have characteristic morphological differences from diploids
(Lewis 1980). The morphological variation observed in the Lupinus latifolius common garden study may
result directly from polyploidy.
If Lupinus latifolius morphological variation is due to polyploidy, should the variants be given a
formal taxonomic name? Autopolyploids are often not separated from diploids taxonomically, though
the decision is made on a case by case basis (Lewis 1980). If two series of populations differing in
chromosome number fail to interbreed, if they also differ consistently in morphology, habitat, and range,
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and if each arose only once, these series of populations should be recognized as two different species.
Not every species meets all of these standards, of course. The diploid and tetraploid populations of
Lupinus latifolius may meet few of them. There are several possible problems.
First, tetraploid Lupinus latifolius may have arisen more than once from its diploid ancestors.
Therefore, a particular tetraploid population's closest relatives may be diploids, rather than other
tetraploid populations. This does not preclude considering diploid and tetraploid L. latifolius as two
species; recurring polyploids can be named. However, it is a problem. The hypothesis that L. latifolius
tetraploids have originated repeatedly can be tested by isozyme analysis of both diploid and tetraploid
populations occurring at distant locations.
Second, irregularities in meiosis and fertilization may facilitate gene flow between diploids and
tetraploids. Diploids may produce unreduced (diploid) pollen or ovules which (in mixed populations)
may fertilize or be fertilized by the gametes of tetraploids. If so, diploids produce some tetraploid
offspring. Also, the normal haploid ovules of tetraploids are functionally diploid. If they are pollinated in
such a way that endosperm is produced but the ovule is not fertilized, the unfertilized diploid ovule may
develop into a diploid seedling.
There is evidence that reproductive barriers between diploid and tetraploid Lupinus latifolius exist but
may be porous. Diploids and tetraploids are genetically differentiated (Fpt > 0.15; Table 10). In all
analyses based on genetic distances, populations 1, 7, and 8, each with several tetraploid individuals,
cluster with each other, not with diploids (Figures 1 - 3). However, population 6, which clustered with
diploids, had three individuals which appeared to have the unbalanced heterozygous genotypes
characteristic of tetraploids. Diploid and tetraploid populations share most alleles, and the alleles unique
to one group are rare. If two groups of populations represent two different species, fixed (consistent)
isozyme differences between them are usually observed, but there were no fixed differences between the
diploid and tetraploid L. latifolius populations observed in this study. Genetic distances among
populations within a species are usually greater than 0.9 (Crawford 1989), and genetic distances among
sampled L. latifolius populations vary from 0.88 to 0.995 (Table 8).
A literature search might well reveal direct experimental evidence of the success or failure of cross
pollination between diploid and tetraploid legumes. In addition, the question could be addressed with
Lupinus latifolius now under cultivation.
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Portland, Oregon.
Doede, David L., Caitlin Cray, Joan Trindle, and Dale Darris. 1998.
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Gottlieb, L. D. 1981. Gene number in species of Asteraceae that have
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different chromosome numbers. Proc. Nat. Acad. Sci. USA 78: 3726-3729
Gottlieb, L. D. 1982. Conservation and duplication of isozymes in
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Gitzendanner, M. A., and P. S. Soltis. 2000. Patterns of genetic variation
in rare and widespread plant congeners. American Journal of
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Gottlieb, L. D. 1981. Gene number in species of Asteraceae that have
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Hamrick, J. L., and M. J. W. Godt. 1990. Allozyme diversity in plant
species. Pages 43-63 In Brown, A. H. D., M. T. Clegg, A. L. Kahler, and B. S. Weir (editors)
Plant Population Genetics, Breeding, and Genetic Resources. Sinauer Associates, Inc.,
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Hamrick, J. L., M. J. W. Godt, D. A. Murawski, and M. D. Loveless.
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implications for conservation biology. Pages 75-86 In Falk, D. A.,
and K. E. Holsinger (editors) Genetics and Conservation of Rare
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Lack of genetic diversity within and among populations of an
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275-282.
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methods for estimating average levels of gene flow. Evolution 43: 1349-1368.
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Laboratory Standard Operating Procedures. NFGEL, USDA Forest Service, Camino, California.
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Wendel, J. F., and N. F. Weeden. 1989. Visualization and interpretation of
plant isozymes. pp. 5-45 in D. E. Soltis and P. S. Soltis, eds. Isozymes in Plant Biology.
Dioscorides Press, Portland, Oregon.
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ACKNOWLEDGEMENTS
We thank Suellen Carroll, Patricia Guge, and Randy Meyer of the National Forest Genetic
Electrophoresis Laboratory for technical support.
This report was prepared by Barbara Wilson and Valerie Hipkins, and are the results of NFGEL
Project #72.
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T8
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Fig1
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NFGEL Staff Activities
Staff Activities
Meetings, Shortcourses, and Workshops
Presentations
2000. V Hipkins. Issues of genetic purity and diversity for
land managers. Native and Invasive Plant Conference, Ft. Collins, CO. November
29 - December 1.
2001. V. Hipkins. Gene conservation and genetic markers
in the USDA Forest Service: From conifers to grasses. Western Forest Genetics
Association. University of California, Davis, July 30 - August 2.
Posters
2001. Conservation Genetics - A History of NFGEL
Projects. Western Forest Genetics Association. University of California, Davis, July
30 - August 2.
Attended
2001. Western Forest Genetics Association. University of
California, Davis, July 30 - August 2. (R.Saich, S.Carroll, P.Guge).
2001. Southern Forest Tree Improvement Conference,
Athens, GA, June 27 - 29. (V. Hipkins)
2001. Budget and Allocation Meeting. USDA Forest
Service, Washington Office, Washington D.C., June 24 - 25. (V. Hipkins)
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NFGEL Staff Activities
Professional Activities
Chen, Z., T.E. Kolb, K.M. Clancy, V.D. Hipkins, and L.E.
DeWald. 2001. Allozyme variation in interior Douglas-fir: association with growth and
resistance to western spruce budworm herbivory. Canadian Journal of Forest Research
31(10):1691-1700.
Wilson, B.L., J. Kitzmiller, W. Rolle, and V.D. Hipkins. 2001.
Isozyme variation and its environmental correlates in Elymus glaucus from the
California Floristic Province. Canadian Journal of Botany 79:139-153.
Samman, S., B.L. Wilson, and V.D. Hipkins. 2001. Genetic
variation in Pinus ponderosa, Purshia tridentata, and Festuca idahoensis,
community-dominant plants of California's yellow pine forest. Madrono 47(3):164-173.
Hipkins, V. 2001. NFGEL reaches milestone - its 100th project.
Eldorado National Forests 'News Nuggets', June, and the Eldorado National Forest
Interpretive Associations 'The Interpreter', September.
Peer reviewer for Canadian Journal of Forest Research, TAG,
and Silvae Genetica (V.Hipkins).
Adjunct faculty member at Northern Arizona University, School
of Forestry, Flagstaff, AZ (V.Hipkins).
Western Forest Genetics Association Officer - Vice Chair
(third year) (V.Hipkins).
Organization Committee for the 2001 Western Forest Genetics
Association Meeting (V. Hipkins)
Participated in hosting the Institute of Forest Genetics 75th
Anniversary Celebration, August 1, 2001 (R. Meyer, B. Carroll, P. Guge, S. Carroll, R.
Saich)
2001 Western Forest Genetics Association conference web
page;development and maintainance(R. Saich and V. Hipkins)
Developed NFGEL website
(http://dendrome.ucdavis.edu/NFGEL/)
(V. Hipkins and R. Saich)
Internal Activities
Member of Region 2,4,5, & 6 FFIS Fire Payment Team (S.Carroll). Member of the
Eldorado National Forest Safety Committee (R.Meyer). Union Representative - Eldorado
National Forest (R.Meyer). Participated in PSW Station Review (V. Hipkins)
Hosted
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NFGEL Staff Activities
NFGEL continues to host a variety of visitors. Tours of the facility and operation were
provided to (1) Forest Service employees representing the Research branch, the
Washington Office, and three Regions of the National Forest System, (2) members of the
public, both from within and outside of California, (3) private industry, (4) university
faculty, (5) foreign scientists from Korea, Poland, Australia, Mexico and Canada, and (6)
employees from other state and federal government agencies. NFGEL hosted groups from
Modesto Jr. College and the Western Forest Genetics Conference, and a graduate student
from Northern Arizona University.
Collaborations and Cooperations
NFGEL formed collaborations with FS Research Stations, Northern Arizona University
(Flagstaff), Bureau of Land Management, California Department of Transportation, US
Fish and Wildlife Service, University of California at Davis, and private companies. We
also collaborate internally within the Agency to lend expertise in the area of genetics.
This took the form of reviewing contracted work reports on the genetics of mammals and
plants.
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NFGEL Staff Activities
Current Staffing
Name
Valerie Hipkins
Suellen Carroll
Pat Guge
Randy Meyer
Robert Saich
Barbara Wilson
Brady Carroll
Position
Director
Lab Manager
Lab Biotechnician
Lab Biotechnician
Lab Biotechnician
Associate Director
Lab Biotechnician
Term
PFT
PFT
PFT
PFT
Temp (10/00 - 10/01)
Temp (8/01 - 10/01)
Temp (6/01 - 8/01)
http://dendrome.ucdavis.edu/NFGEL/NFGEL01/staff.html [8/1/2003 9:32:44 AM]
E-mail Adress
vhipkins@fs.fed.us
scarroll@fs.fed.us
pguge@fs.fed.us
rmeyer@fs.fed.us
rcsaich@fs.fed.us
blwilson@fs.fed.us
NFGEL Budget 2000-2001
Budget
Activity
FY98
FY99
FY00
FY01
Receipts (in thousands)
Allocation
Carryover
Soft Money
Total
290.0
0.0
22.7
307.0
9.9
25.8
290.0
0.2
26.5
290.0
6.9
169.8
312.7
342.7
316.7
466.7
142.9
29.5
67.3
0.0
26.4
22.9
5.1
4.4
1.5
0.9
0.6
0.5
0.4
0.4
0.0
0.0
0.0
165.3
58.5
69.0
0.0
16.5
10.1
8.6
4.4
0.5
0.8
4.9
0.3
0.5
0.4
0.8
1.5
0.0
171.5*
25.6
59.0
0.0
23.6
3.2
11.6
0.0
0.5
1.0
3.7
1.2
1.2
0.9
0.2
0.4
0.0
185.7**
19.2
60.0
10.9
15.7
164.2
5.0
2.5
0.3
0.0
0.8
0.8
1.2
0.1
0.1
0.0
9.7
302.8
342.1
Expenditures (in thousands)
Salary (permanant)
(temperary)
Overhead to ENF
Overhead to P'ville Nursery
Chemicals/Supplies
Equipment
Travel/Training
Awards
Fees
Books/subscriptions
Computers (not including FOR)
Repair
Photos/Slides/Publications
Postage
Office Supplies
Furniture
Lab Relocation
Total
303.6
*$5.2 salary savings due to alternate salary sources
**$11.7 salary savings due to alternate salary sources
FY 01 Soft Money
Source
FS-NFP (RMRS)
FS-NFP (WO)
FS-SRS
FS-R6
FS-R4
Amount ($)
(Native Plants)
(Equipment)
(Southern pines)
(Aspen)
(Lewisia)
Total
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Percentage
72,254
85,000
5,000
2,500
5,000
42.6%
50.1%
2.9%
1.5%
2.9%
169,754
100.0%
476.2
NFGEL Workload By Project
NFGEL Projects (2001)
Project# Collaborator
74
94
99
Teresa
Prendusi,
FS-R4
Species
Objective
Lewisia
kelloggii
(1) Assess
levels and
structure of
genetic
variation in and
among
populations of
Lewisia
kelloggii
growing in
Idaho, and (2)
determine
genetic
similarity
between Idaho
and California
populations of
L. kelloggii
Sample
Type
leaves
Sample Submission Preparation Electrophoresis
Size
Dates
Dates
Dates
385
indiv.
Nick Wheeler,
Pseudotsuga
Weyerhaeuser
menziesii
Co.
To determine
the efficancy of
5 indiv.,
supplemental
6
seed
mass
(meg/embryo seedlots
pollination
(328
pairs)
methods in a
seed)
Douglas-fir
seed orchard
Kristin
Kolanowski,
NAU
(1) Determine
the genetic
variation
partitioning of
clumps of trees
that
established
prior to
Euro-American
settlement
(1876),(2)
determine the
genetic
composition
and structure
of pre- and
post
-settlement
tree,(3)
compare
allozyme
variation of the
two age
groups, and(4)
compare the
genetic
composition
and structure
of 5 stands
prior to and
after simulated
random
thinnings
having
50,25,and 10%
post-settlement
retaining
percentages
Pinus
ponderosa
vegetative
buds
465
indiv.
Marker
System
#Loci
6/99 - 6/00
7/99 - 6/00
10/00 - 12/00
isozymes
19
3/00, 12/00
3/00, 12/00
- 2/01
3/00, 12/00
-2/01
isozymes
15
2/01
2/01
2/01
isozymes
22
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NFGEL Workload By Project
98
Lisa Schicker,
CalTrans
Durant
104-119 MacArthur,
FS-RMRS
112
Bryan Schulz,
Olympic
Resource
Management
Pinus radiata
(1) Did the
planted
Monterey pine
trees along
Highway 1 in
Monterey
county near
Carmel
originate from
one of the
three natural
mainland P.
radiata
populations
(Monterey,
Cambria, or
Ano Nuevo)?
(2) How
genetically
similar is the
Hatton Canyon
stand relative
to other stands
in the
Monterey
population? (3)
Is overall
genetic
variation
reduced in
selected pitch
canker
resistant
material
compared to
the species as
a whole, or
compared to
susceptible
material?
(Contingent on
common
garden
material being
available at a
latter date for
genetic
testing).
Bromus
carinatus,
Viguiera
multiflora,
Astragalus
utahensis,
Crepis
acuminata,
Eriogonum
umbellatum,
Lupinus
argenteus,
Erigeron
pumilus, Vicia
americana
Pseudotsuga
menziesii
seed (megs)
254
indiv.
1/01
2/01 - 3/01
3/01 - 8/01
isozymes
26
Methods
development
leaves
--
5/01 - 7/01
5/01 - 7/01
5/01 - 7/01
isozymes
--
Clonal
identification.
Progeny to
parent
identification.
vegetative
buds
44 indiv.
6/01
6/01
8/01
isozymes
18
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NFGEL Workload By Project
102
97
102
105
Richard
Sniezko,
FS-R6
Chamaecyparis
lawsoniana,
Chamaecyparis
obtusa,
Chamaecyparis
nootkatensis,
Chamaecyparis
thyoides
Verify hybrid
seedlings
between
CHLA, CHNO,
CHOB, and
CHTH
needles
52 indiv.
5/01
3/01
8/01 - 9/01
isozymes
19
Richard
Sniezko,
FS-R6
Chamaecyparis
lawsoniana,
Ch. obtusa,
Ch. leylandii,
Ch.
Ramet/hybrid
nootkantensis, identification
Ch. thyoides,
Cupressus
macrocarpa, C.
tortulosa
needles
34
indiv.,
45
primers
3/00 - 5/00
--
10/00 - 12/00
RAPDs
--
Richard
Sniezko,
FS-R6
Chamaecyparis
lawsoniana,
Chamaecyparis
obtusa,
Chamaecyparis
nootkatensis,
Chamaecyparis
thyoides
Verify hybrid
seedlings
between
CHLA, CHNO,
CHOB, and
CHTH
needles
54 indiv.
5/01
6/01
--
DNA
Extraction
--
Viguiera
multiflora
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
53 indiv.
5/01
5/01 - 8/01
--
DNA
Extraction
--
Durant
MacArthur,
FS-RMRS
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NFGEL Workload By Project
107
109
Durant
MacArthur,
FS- RMRS
Durant
MacArthur,
FS- RMRS
Erigeron
pumilus
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
65 indiv.
5/01
6/01 - 8/01
--
DNA
Extraction
--
Crepis
acuminata
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
49 indiv.
5/01
6/01 -8/01
--
DNA
Extraction
--
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NFGEL Workload By Project
111
114
Durant
MacArthur,
FS- RMRS
Durant
MacArthur,
FS- RMRS
Astragalus
utahensis
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
32 indiv.
5/01
6/01 - 8/01
--
DNA
Extraction
--
Eriogonum
umbellatum
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
63 indiv.
6/01
6/01 - 8/01`
--
DNA
Extraction
--
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NFGEL Workload By Project
116
118
Durant
MacArthur,
FS- RMRS
Durant
MacArthur,
FS- RMRS
Lupinus
argenteus, L.
sericeus
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
67 indiv.
7/01
7/01- 8/01
--
DNA
Extraction
--
Bromus
carinatus
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
leaves
22 indiv.
7/01 - 8/01
7/01 - 8/01
--
DNA
Extraction
--
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NFGEL Workload By Project
120
Durant
MacArthur,
FS- RMRS
To examine (1)
gene flow and
genetic
population
structure of
source
populations,
seeded
populations,
and indigenous
populations
adjacent to the
seeded
populations,
Vica americana
and (2) the
intrapopulation
and
interpopulation
genetic
structure and
variation of
material being
introduced to
revegation and
restoration
treatments of
disturbed
wildlands.
103
Mary Frances
Mahalovich,
FS-R1
Pinus
ponderosa
Source
identification
leaves
41 indiv
7/01 - 8/01
8/01
--
DNA
Extraction
--
needles
158
indiv.
5/01 - 9/01
6/01- 8/01
--
DNA
extraction
--
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Workload by Region or Agency, FY01
(1) Isozymes (starch gel electrophoresis)
By Project
Region or Agency
Forest Service
R4
R3/NAU
R6/BLM
RMRS
NFGEL
CalTrans
Weyerhaeuser
Olympic
Resource Mgt.
Project #
74
99
102
104-119
-98
94
112
Species
# gels
# days
# weeks
Lewisia kelloggii
Ponderosa pine
Chamaecyparis
development
development
Monterey pine
Douglas fir
75
63
12
12
6
153
54
20
9
2
1
1
24
10
10.0
3.0
1.0
0.5
0.5
15.0
6.0
Douglas fir
14
3
1.5
By Forest Service Region or Agency
Region or Agency
# gels
# days
# weeks
Forest Service
NFS
R4
R3/NAU
R6/BLM
NFGEL
75
63
12
6
20
9
2
1
10.0
3
1
0.5
RMRS
CalTrans
Weyerhaeuser Co
Olympic
Resource Mgt
12
153
54
1
24
10
0.5
15.0
6.0
14
3
1.5
TOTAL
389
70
37.5
FSR
(2) DNA
Region or
Agency
Project #
species
# DNA
extractions
Extraction
Method
# PCR reactions # days
# weeks
FS-NFS-R6 / BLM
97
Chamaecyparis lawsoniana,
Ch. obtusa,
Ch. leylandii,
Ch. nootkantensis, Ch. thyoides,
Cupressus macrocarpa,
C. tortulosa
FS-NFS-R1
103
Pinus ponderosa
158
FastPrep
--
20
7
FS-FSR-RMRS
105
Viguiera multiflora
53
FastPrep
--
30*
13*
FS-FSR-RMRS
107
Erigeron pumilus
65
FastPrep
--
30*
13*
FS-FSR-RMRS
109
Crepis acuminata
49
FastPrep
--
30*
13*
FS-FSR-RMRS
111
Astragalus utahensis
32
FastPrep
--
30*
13*
FS-FSR-RMRS
114
Eriogonum umbellatum
63
FastPrep
--
30*
13*
34
FastPrep
21
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27
8
FS-FSR-RMRS
116
Lupinus argenteus, L. sericeus
67
FastPrep
--
30*
13*
FS-FSR-RMRS
118
Bromus carinatus
22
FastPrep
--
30*
13*
FS-FSR-RMRS
120
Vica americana
41
FastPrep
--
30*
13*
BLM=Bureau of Land Management
NAU=Northern Arizona University
CalTrans=California Department of Transportation
FS=Forest Service
FSR=Forest Service Research
RMRS=Rocky Mountain Research Station
NFS=National Forest System
R#=Region
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